WO2010065626A1

WO2010065626A1 - Genotyping tools, methods and kits

Info

Publication number: WO2010065626A1
Application number: PCT/US2009/066393
Authority: WO
Inventors: Yingnian Lu; Amber E. Britton; Thomas H. King; Alex Franzusoff; Zhimin Guo
Original assignee: Globeimmune, Inc.
Priority date: 2008-12-02
Filing date: 2009-12-02
Publication date: 2010-06-10

Abstract

Described are products, kits and methods useful for amplifying and detecting rare mutations, including probes, primers and PNA useful for amplifying and detecting rare mutations, and a method and kit for detecting one or more mutations in a gene. The invention detects rare mutations or those that are present in a small percentage of total cells within a diseased tissue.

Description

GENOTYPING TOOLS, METHODS AND KITS

Reference to Sequence Listing

This application contains a Sequence Listing submitted electronically as a text file by EFS-Web. The text file, named "3923-22-PCT_ST25", has a size in bytes of 24KB, and was recorded on 1 December 2009. The information contained in the text file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

Field of the Invention

This invention generally relates to products, kits and methods useful for amplifying and detecting rare mutations, including probes, primers and PNA useful for amplifying and detecting rare mutations, and a method and kit for detecting one or more mutations in a gene. The invention is particularly useful for detecting rare mutations or those that are present in a small percentage of total cells within a diseased tissue.

Background of the Invention Personalized therapy is becoming a reality in the treatment of many cancers and other diseases. Therapy options for cancer, for example, have expanded considerably over recent years, and include a variety of chemotherapeutic drugs and combinations thereof, as well as new targeted therapies, such as immunotherapeutic cancer therapy. With the expanding options for treatment, however, comes an increased possibility of toxicities, non-responsiveness, and treatment costs, making the identification of tools that can select the best therapy for a given patient an important factor in prescribing treatment. Indeed, some therapeutic approaches may be largely or completely ineffective for treating a particular individual, rendering the use of such therapy on that patient costly both in terms of patient health and actual expense, not to mention that it may deny the patient the opportunity to use a more effective therapy. Moreover, some of the aforementioned targeted therapies for cancer and other diseases may be specific for a particular patient genotype, therefore requiring the initial identification of the patient's genotype prior to drug administration.

By way of example, it has recently been demonstrated that epidermal growth factor receptor (EGFR) inhibitors, useful for treating colorectal and other cancers, do not appear to work in patients harboring certain activating K-ras mutations. More specifically, data pooled from four clinical trials confirmed that, in colorectal cancer patients receiving the

EGFR inhibitor panitumumab (Vectibix®; Amgen), responses to the panitumumab were observed only in patients with wild-type K-Ras status; patients having mutations in K-Ras failed to respond to the drug (ASCO-NCI-EORTC Annual Meeting on Molecular markers in Cancer, October 31, 2008). Similarly, another study has shown that patients "with a colorectal tumor bearing mutated K-ras did not benefit from" another EGFR inhibitor, cetuximab, whereas patients with tumors having wild-type K-ras did benefit from the drug (Karapetis et al, 2008, N. Engl. J. Med. 359:1757-1765). Accordingly, K-Ras status in cancer patients should now be used as a predictor of patients who are likely to benefit from EGFR inhibitor therapy, where patients with K-Ras mutations should be offered a different type of treatment. This concept may well apply to as yet to be identified cancer drugs, especially those that target proteins that do not drive the disease.

In addition to screening patients for point mutations, the ability to screen patients for chromosomal rearrangements that have generated new junctional DNA sequences (e.g., the Bcr-Abl fusion in chronic myelogenous leukemia) is also desirable.

Beyond these cancer applications, the personalized therapy concept extends to infectious diseases. For example, the efficiency of replication for certain viral diseases such as Hepatitis B virus (HBV), Hepatitis C Virus (HCV) and Human Immunodeficiency Virus- type 1 (HIV-I) depends upon a variety of factors including: 1) host genetic makeup and frequency of certain mutations (Jopling and Norman, 2006; Yu et al., 2007, J. Virol., 81(4):1619-1631), and; 2) the presence of 'escape mutations' in the genomes of the viruses themselves which render the viruses refractory to drug efficacy or recognition by the immune system (Wolfl et al., 2008, J. Immunol. 181(9):6435-6446; Shibata et al., 2007, J. Virol, 81(8):3757-3768; Sloan et al., 2008, Antivir. Ther. 13(3):439-447). These facts underscore the need for rapid, sensitive, and high throughput detection systems that can detect mutations in both host and pathogen genes. In addition, there is a need in the art to genotype fetal DNA, which is currently limited by access to the fetal DNA and/or limited available quantities of such DNA.

In general, diagnostics that are designed to be used in connection with therapeutic approaches for patient treatment are not widely available. Indeed, there are currently no FDA approved diagnostics, devices or test systems commercially available for the analysis of the Ras gene, including H-Ras, N-Ras or K-Ras. Therefore, there is a need in the art for new methods and products for genotyping patients, particularly for rare or those that are present in a small percentage of total cells within a given tissue, such as a diseased tissue (underrepresented mutations). In addition, given that the amount and/or quality of a biological sample available for screening from a given patient may be limited, there is a need for genotyping methods and products that are efficient and sensitive, and that can maximize the information obtainable from patient genetic material.

Summary of the Invention One embodiment of the invention relates to oligonucleotide primers. Also included in this embodiment of the invention is a composition or a kit comprising one or more of such oligonucleotide primers. The oligonucleotide primers are useful, in one aspect, for amplifying one or more target polynucleotides from a nucleic acid sample to produce at least one amplification product, each amplification product containing a selected site of a target polynucleotide. In one aspect, the target polynucleotide is from a gene selected from ras, braf, or egfr {i.e., genes encoding Ras, B-Raf, or EGFR, respectively).

In one aspect, the invention includes an oligonucleotide primer that hybridizes to, and/or is used for amplification of, sequences from exon 3 of ras and which include codon 61 of ras. These primers are selected from a primer having a sequence including or consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:59, or SEQ ID NO: 60. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above -identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, the combination of SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:27 and SEQ ID NO:30, SEQ ID NO:27 and SEQ ID NO:48, SEQ ID NO:27 and SEQ ID NO:56, SEQ ID NO:27 and SEQ ID NO:60, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:29 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:48, SEQ ID NO:29 and SEQ ID NO:56, SEQ ID NO:29 and SEQ ID NO:60, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:47 and SEQ ID NO:56, SEQ ID NO:47 and SEQ ID NO:60, SEQ ID NO:47 and SEQ ID NO:28, SEQ ID NO:47 and SEQ ID NO:30, SEQ ID NO:55 and SEQ ID NO:56, SEQ ID NO:55 and SEQ ID NO:48, SEQ ID NO:55 and SEQ ID NO:60, SEQ ID NO:55 and SEQ ID NO:28, SEQ ID NO:55 and SEQ ID NO:30, SEQ ID NO:59 and SEQ ID NO:60, SEQ ID NO:59 and SEQ ID NO:48, SEQ ID NO:59 and SEQ ID NO:56, SEQ ID NO:59 and SEQ ID NO:28, or SEQ ID NO:59 and SEQ ID NO:30.

In one aspect, the invention includes an oligonucleotide primer that hybridizes to, and/or is used for amplification of, sequences from exon 2 of ras, and which include codon 12 of ras. These primers are selected from a primer having a sequence including or consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, or SEQ ID NO:58. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above-identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, SEQ ID NO:7 with any of SEQ ID NO:8, SEQ ID NO: 10 or SEQ ID NO: 11; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:53 and SEQ ID NO:54, or SEQ ID NO:57 and SEQ ID NO:58. Any other combination of forward and reverse primers selected from the above primers is also encompassed by the invention.

In one aspect, the invention includes an oligonucleotide primer that hybridizes to, and/or is used for amplification of, sequences that include codon 600 of braf. These primers are selected from a primer having a sequence including or consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:49 or SEQ ID NO:50. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above- identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, SEQ ID NO:36 and SEQ ID NO:37, or SEQ ID NO:49 and SEQ ID NO:50. Any other combination of forward and reverse primers selected from the above primers is also encompassed by the invention.

In one aspect, the invention includes an oligonucleotide primer that hybridizes to, and/or is used for amplification of, sequences from exon 19 of the gene encoding EGFR. These primers are selected from a primer having a sequence including or consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:51 or SEQ ID NO:52. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above -identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, SEQ ID NO:41 and SEQ ID NO:42 or SEQ ID NO:51 and SEQ ID NO:52. Any other combination of forward and reverse primers selected from the above primers is also encompassed by the invention.

Another embodiment of the invention relates to a method for amplifying a target polynucleotide, comprising amplifying a target polynucleotide from a nucleic acid sample to produce an amplification product, wherein the amplification is performed using a pair of oligonucleotide primers. In this embodiment, at least one of the primers is an oligonucleotide primer selected from SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:51 or SEQ ID NO:52. Various combinations of primer pairs are described above and are encompassed by this embodiment of the invention.

Yet another embodiment of the present invention relates to PNA molecules. Also included in this embodiment of the invention is a composition or a kit comprising one or more of such PNA molecules. The PNA molecules are useful, in one aspect, for inhibition of the amplification of target polynucleotides having a non-target sequence at a selected site, thereby enhancing amplification of target polynucleotides having a target sequence at the selected site. In one aspect, the target polynucleotide is from a gene selected from ras, b-raf, or the gene encoding EGFR (i.e., genes encoding Ras, B-Raf, or EGFR, respectively). In one aspect of the invention, a PNA is used in conjunction with amplification primers that overlap with at least a portion of the polynucleotide to which the PNA binds.

PNA useful for the inhibition of the amplification of a portion of wild-type ras comprising exon 3, including codon 61, includes a PNA that includes or consists of the sequence of: SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type ras in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds. For example, in one aspect, the PNA of SEQ ID NO:26 inhibits amplification of wild-type ras in an amplification reaction in which SEQ ID NO:29 and/or SEQ ID NO:30 are used.

PNA useful for the inhibition of the amplification of a portion of wild-type ras comprising exon 2, including codon 12, includes a PNA that includes or consists of the sequence of SEQ ID NO: 17. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type ras in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds.

PNA useful for the inhibition of the amplification of a portion of wild-type braf comprising codon 600 includes a PNA that includes or consists of the sequence of SEQ ID NO:38. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type braf in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds.

PNA useful for the inhibition of the amplification of a portion of wild-type egfr includes a PNA that includes or consists of the sequence of SEQ ID NO:43. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type egfr in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds.

Another embodiment of the invention relates to a method for inhibiting the amplification of a non-target sequence, comprising amplifying a target polynucleotide in the presence of a PNA selected from SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,

SEQ ID NO:26, SEQ ID NO: 17, SEQ ID NO:38 or SEQ ID NO:43. In one aspect of the invention, the PNA is used in conjunction with amplification primers that overlap with at least a portion of the nucleotide sequence to which the PNA binds. For example, the PNA of SEQ ID NO:26 can be used in a method of amplifying wherein primers of SEQ ID NO:29 and/or SEQ ID NO:30 are used.

Yet another embodiment of the present invention relates to oligonucleotide probes. Also included in this embodiment of the invention is a composition or a kit comprising one or more of such oligonucleotide probes. The oligonucleotide probes are useful, in one aspect, for hybridization to a target sequence in a target polynucleotide, such as for detection of target sequences in a nucleic acid sample. In one aspect, the target polynucleotide is from a gene selected from ras, b-raf or egfr (i.e., genes encoding Ras, B-Raf, or EGFR, respectively).

Probes useful for the hybridization to and detection of a target sequence within exon 3 of ras, which may include wild-type and/or mutated ras sequences and include codon 61, include probes with a sequence including or consisting of: SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35.

Probes useful for the hybridization to and detection of a target sequence within exon 2 of ras, which may include wild-type and/or mutated ras sequences and include codon 12, include probes with a sequence including or consisting of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

Probes useful for the hybridization to and detection of a target sequence within braf, which may include wild-type and/or mutated braf sequences, include probes with a sequence including or consisting of: SEQ ID NO:39 or SEQ ID NO:40.

Probes useful for the hybridization to and detection of a target sequence within exon 19 of the EGFR gene, which may include wild-type and/or mutated sequences, include probes with a sequence including or consisting of: SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46. Another embodiment of the invention relates to a method for detecting a polynucleotide sequence comprising contacting a polynucleotide with an oligonucleotide probe selected from SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46, and detecting whether the probe hybridizes to the polynucleotide sequence. In one aspect, the probe can be labeled. In another aspect, the probe is not labeled.

One embodiment of the invention relates to a method for detecting target sequences in a nucleic acid sample. The method includes the steps of: (a) amplifying one or more target polynucleotides from a nucleic acid sample to produce at least one amplification product, each amplification product containing a selected site of a target polynucleotide for detection of target sequences; (b) isolating single-stranded polynucleotides from the amplification product; (c) contacting the single stranded polynucleotides with a hybridization probe and with a label that detects hybridization of a single-stranded polynucleotide and the hybridization probe, under conditions sufficient to cause the single-stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide; (d) detecting the melting temperature (T_m) of the hybridized polynucleotide; and (e) detecting target sequences at the selected site of the target polynucleotides by detecting perfectly hybridized polynucleotides, wherein perfectly hybridized polynucleotides have a higher T_m than hybridized polynucleotides formed with a probe having a nucleic acid sequence that is different from the single-stranded polynucleotide at the selected site by at least one nucleotide. During the amplification step (a), amplification of target polynucleotides having a non-target sequence at the selected site is inhibited, thereby enhancing amplification of target polynucleotides having a target sequence at the selected site. The hybridization probe(s) used in step (c) is configured to hybridize to a nucleic acid sequence spanning the selected site of the target polynucleotide, and has a nucleic acid sequence at the selected site corresponding to either (i) a target sequence, or (ii) a non-target sequence. Any of the various aspects and embodiments of the method of the invention described herein, including below, can be used together or as alternates, in any combination, as is reasonable and/or desired for performance of the method.

In one aspect of the method of the invention, step (a) is performed in the presence of a nucleic acid binding moiety that binds to a non-target sequence at the selected site and thereby inhibits the amplification of a target polynucleotide having the non-target sequence at the selected site. For example, such a nucleic acid binding moiety can include, but is not limited to, a peptide nucleic acid (PNA).

In one aspect of the method, step (a) is performed using polymerase chain reaction (PCR). For example, step (a) can include a single PCR, or in an alternate embodiment, a first and a second PCR. In the latter embodiment, in one aspect, the amplification of the target polynucleotide comprising a non-target sequence is inhibited during the first PCR and optionally during the second PCR. In one aspect, the PCR is performed using a pair of oligonucleotide primers for amplifying the target polynucleotide, wherein either primer comprises a selection moiety for isolating single-stranded polynucleotides from the amplification product. Such a selection moiety can include, but is not limited to, biotin. In an example where biotin is the selection moiety, step (b) can include binding the amplification product to streptavidin immobilized on a substrate, followed by denaturing the amplification product to produce a single-stranded polynucleotide that is bound to the substrate. In another aspect, step (b) comprises binding the amplification product to an immobilized substrate, followed by denaturing the amplification product into single- stranded polynucleotides. In any of these aspects of the invention, the substrate can include, but is not limited to, a magnetic bead. In one aspect, such a substrate does not substantially interfere with the detection of the detectable label used in step (c).

In one aspect of the method of the invention, the step (b) of isolating single- stranded polynucleotides from the amplification product includes exposing the amplification product to denaturing conditions. Such denaturing conditions may include, but are not limited to, exposure of the amplification product to conditions including 0.15M NaOH. In one aspect of this embodiment of the invention, the amplification product is between about 45 and about 1000 nucleotides in length. In another aspect, the amplification product is between about 45 and about 500 nucleotides in length. In another aspect, the amplification product is less than about 300 nucleotides in length. In another aspect, the amplification product is less than about 100 nucleotides in length. In another aspect, the amplification product is less than about 80 nucleotides in length. In yet another aspect, the amplification product is about 70 to about 80 nucleotides in length.

In one aspect, the amplification product is purified from other PCR components between steps (a) and (b). In one aspect of the method of the invention, the label in step (c) is a double stranded nucleotide binding agent. For example, the double stranded nucleotide binding agent can include, but is not limited to an intercalating agent, such as a fluorescent dye.

In one aspect, step (c) of the method of the invention comprises distributing the single-stranded polynucleotides from step (b) into two or more aliquots, wherein each aliquot is contacted with the label and one of a plurality of hybridization probes to form a hybridized polynucleotide, each hybridization probe in the plurality having a different sequence at the selected site as compared to the other probes in the plurality; and wherein each aliquot is contacted with a different hybridization probe. In one further aspect, step (d) comprises determining the T_m for each of the hybridized polynucleotides, and wherein step (e) comprises comparing the T_m from the hybridized polynucleotides in each aliquot to the T_m from the hybridized polynucleotides in the other aliquots, wherein hybridized polynucleotides having a T_m that is significantly higher than the others is identified as including a probe that is fully complementary to the single-stranded polynucleotide, thereby identifying the target sequence or lack thereof at the selected site of the target polynucleotide.

In another aspect of the method of the invention, step (c) comprises distributing the single-stranded polynucleotides from step (b) into two or more aliquots, wherein each aliquot is contacted with the label and two or more of a plurality of hybridization probes to form the hybridized polynucleotides, each hybridization probe in the plurality and in the aliquot having a different sequence at the selected site as compared to the other probes in the plurality and in the aliquot.

In one aspect of the method of the invention, step (c) is conducted in between about 0.1X and about 0.5X SSC. In one aspect of the invention, the hybridization probe is between about 10 and about 30 nucleotides in length. In another aspect, the hybridization probe is less than about 20 nucleotides in length. In yet another aspect, the hybridization probe is about 15 nucleotides in length. In one aspect of the invention, the nucleic acid sample is DNA extracted from a patient biological sample. In one aspect, the nucleic acid sample is DNA extracted from a patient tumor sample. In one aspect, the nucleic acid sample used in step (a) comprises between about 20 ng and about 1 mg of patient DNA. In another aspect, the nucleic acid sample used in step (a) comprises at least about 20 ng of patient DNA. In one aspect of the invention, the single-stranded polynucleotide used in step (c) comprises between about 20 ng and about 300 ng of polynucleotide.

In one aspect of the invention, the target sequence includes a substitution of at least one nucleotide at the selected site for a different nucleotide as compared to a non-target sequence. In one aspect, the substitution is a point mutation. In another aspect, the target sequence includes a deletion of nucleotides as compared to a non-target sequence. In one aspect, the target polynucleotide is at least a portion of a ras gene. In one aspect, the ras gene is K-ras. In one aspect, the selected site spans a codon of ras selected from the group consisting of 12, 13, 59, 61 and 76. In one aspect, the target nucleotide is at least a portion of a gene encoding BRAF. In one aspect, the target nucleotide is at least a portion of an epidermal growth factor receptor (EGFR) gene. In another aspect, the method detects at least two different target sequences from the same gene. In one aspect, the method detects target sequences from at least two different selected sites in the same gene. In one aspect, the method detects target sequences from at least two different genes. In one aspect, the method detects target sequences from a virus. In one aspect, the method detects target sequences from a pathogen. In another aspect, the method detects target sequences that are escape mutations from small molecule or immune system therapeutic approaches. In one aspect, the method detects target sequences from two or more genes in the same biological pathway. In one aspect, the target sequence is a mutant sequence associated with a disease or condition, and the method further includes preparing a report for a clinician or other party that identifies the mutation or lack thereof in the target polynucleotide. In one aspect, the target sequence is a mutant sequence associated with a disease or condition, and the method further includes prescribing a mutation-specific treatment to a patient carrying the mutation. Another embodiment of the invention relates to a method of prescribing treatment for a cancer that includes identification of a particular mutation in the DNA of a patient. The method includes the steps of: (a) identifying a mutation in a target polynucleotide of a patient who has cancer by reviewing a report that identifies the mutation, wherein the mutation was detected using any of the methods of the invention described herein; and (b) administering to the patient a therapy that is specific for the mutation identified in the report.

Another embodiment of the invention relates to a method to manufacture a therapeutic agent that is specific for one or more mutations associated with a disease or condition. The method includes the steps of: (a) producing a therapeutic agent that is specific for one or more mutations associated with a disease or condition; and (b) labeling packaging containing the therapeutic agent to require the use of the method as described herein to confirm the presence of the specific mutation or mutations in a patient in conjunction with administration of the agent to the patient. Yet another embodiment of the invention relates to a packaged medicament that is specific for one or more mutations associated with a disease or condition. The medicament includes: (a) a therapeutic agent that is specific for one or more mutations associated with a disease or condition; and (b) package labeling that requires the use of the method described herein to confirm the presence of the specific mutation or mutations in a patient in conjunction with administration of the agent to the patient.

Another embodiment of the invention relates to a kit for detecting target sequences in a nucleic acid sample. The kit includes: (a) at least one pair of PCR primers for producing amplification products from target polynucleotides that contain a selected site; (b) one or more reagents that inhibit the amplification of target polynucleotides having a non-target sequence at the selected site; (c) one or more reagents for isolating single- stranded polynucleotides from the amplification product; (d) a label that detects hybridization of a single-stranded polynucleotide and a hybridization probe; and (e) one or more hybridization probes configured to hybridize to a nucleic acid sequence spanning the selected site of the target polynucleotide, wherein each hybridization probe has a nucleic acid sequence at the selected site corresponding either to (i) a target sequence; or (ii) a non-target sequence. The kit may generally include any one or more agents for use in any of the methods of the invention as described herein.

In one aspect of this embodiment of the invention, at least one of the PCR primers in the pair comprises a selection moiety for isolating single-stranded polynucleotides from the amplification product. For example, such a selection moiety can include, but is not limited to, biotin. In the example of biotin as a selection moiety, the reagent in (c) can include a streptavidin-coated substrate. In one aspect, the reagent in (b) is a nucleic acid binding moiety that binds to a non-target sequence at the selected site and thereby inhibits the amplification of a target polynucleotide having the non-target sequence at the selected site. For example, such a reagent can include, but is not limited to, a peptide nucleic acid (PNA).

In one aspect, the label of (d) is a double stranded nucleotide binding agent. For example, such a double stranded nucleotide binding agent can include, but is not limited to, an intercalating agent. In one aspect, the agent is a fluorescent dye.

In one aspect of this embodiment, the hybridization probe is between about 10 and about 30 nucleotides in length. In another aspect, the hybridization probe is less than about 20 nucleotides in length. In another aspect, the hybridization probe is about 15 nucleotides in length.

In one aspect, the target sequence includes a substitution of at least one nucleotide at the selected site for a different nucleotide as compared to a non-target sequence. For example, such a substitution can include, but is not limited to, a point mutation. In one aspect, the target sequence includes a deletion of nucleotides as compared to a non-target sequence. In one aspect, the target polynucleotide is at least a portion of a ras gene. In one aspect, the ras gene is K-ras. In one aspect, the selected site is within a codon of ras selected from the group consisting of 12, 13, 59, 61 and 76. In one aspect, the target nucleotide is at least a portion of a gene encoding BRAF. In one aspect, the target nucleotide is at least a portion of an epidermal growth factor receptor (EGFR) gene. Another embodiment of the invention relates to a method to manufacture an assay kit for screening for one or more mutations associated with a disease or condition. The method includes the steps of: (a) manufacturing any of the assay kits described herein, wherein the assay kit screens for one or more mutations associated with a disease or condition; and (b) preparing packaging for the kit that includes instructions for the use of the assay kit prior to administration of a specific therapeutic agent that targets one of the mutations that is screened for by the assay kit.

Yet another embodiment of the invention relates to a method for detecting target sequences in a nucleic acid sample. The method includes the steps of: (a) amplifying by polymerase chain reaction (PCR) one or more target polynucleotides from a nucleic acid sample to produce at least one amplification product, each amplification product containing a selected site of a target polynucleotide for detection of target sequences; (b) purifying the amplification product; (c) isolating the amplification product by binding the amplification product to streptavidin conjugated to an immobilized substrate; (d) exposing the bound amplification product to denaturing conditions sufficient to produce a single- stranded polynucleotide bound to the substrate; (e) contacting the single stranded polynucleotides with a hybridization probe and with a double-stranded nucleotide binding agent, under conditions sufficient to cause the single-stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide; (f) detecting the melting temperature (T_m) of the hybridized polynucleotide; and (g) detecting target sequences at the selected site of the target polynucleotides by detecting perfectly hybridized polynucleotides, wherein perfectly hybridized polynucleotides have a higher T_m than hybridized polynucleotides formed with a probe having a nucleic acid sequence that is different from the single-stranded polynucleotide at the selected site by at least one nucleotide. In this embodiment, the step of amplifying is performed in the presence of a peptide nucleic acid (PNA) molecule that binds to a non-target sequence at the selected site and inhibits the amplification of a target polynucleotide having the non-target sequence at the selected site, thereby enhancing amplification of target polynucleotides having a target sequence at the selected site. The PCR is performed using a pair of oligonucleotide primers for amplifying the target polynucleotide, wherein either primer comprises biotin for isolating single-stranded polynucleotides from the amplification product. The hybridization probe is configured to hybridize to a nucleic acid sequence spanning the selected site of the target polynucleotide, and wherein the hybridization probe has a nucleic acid sequence at the selected site corresponding to either (i) a target sequence, or (ii) a non-target sequence.

Another embodiment of the invention relates to a method for detecting mutated ras sequences in a nucleic acid sample. The method includes the steps of: (a) amplifying by polymerase chain reaction (PCR) one or more fragments of a ras gene from a nucleic acid sample to produce at least one amplification product, each amplification product containing a selected site comprising a specific codon of ras for detection of target sequences; (b) purifying the amplification product from other PCR reaction material; (c) isolating the amplification product by binding biotin-labeled polynucleotide to a streptavidin molecule conjugated to a magnetic bead; (d) exposing the bound amplification product to denaturing conditions sufficient to separate the non-biotin-labeled strand of the amplification product from the bound, biotin-labeled strand to produce a single-stranded polynucleotide bound to the bead; (e) contacting the single stranded polynucleotide with a hybridization probe and with a fluorescent double-stranded nucleotide binding agent, under conditions sufficient to cause the single-stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide; (f) detecting the melting temperature (T_m) of the hybridized polynucleotide; and (g) detecting mutated ras sequences at the selected site of the ras fragment by detecting perfectly hybridized polynucleotides, wherein perfectly hybridized polynucleotides have a higher T_m than hybridized polynucleotides formed with a probe having a nucleic acid sequence that is different from the single-stranded polynucleotide at the selected site by at least one nucleotide. The step of amplifying is performed in the presence of a peptide nucleic acid (PNA) molecule that binds to wild-type ras at the selected site and inhibits the amplification of a fragment having a wild-type ras sequence at the selected site, thereby enhancing amplification of ras fragments having a mutated ras sequence at the selected site. The PCR is performed using a pair of oligonucleotide primers for amplifying the ras fragment, wherein one primer in the pair comprises biotin, producing an amplification product having one polynucleotide strand labeled with biotin. The hybridization probe is configured to hybridize to the complement of a nucleic acid sequence spanning the selected site of the ras gene, and wherein the hybridization probe has a nucleic acid sequence at the selected site corresponding to either (i) a mutated ras sequence, or (ii) a wild-type ras sequence.

Any of the methods described above, and corresponding kits or medicaments, can be readily adapted to detect any gene or combination of genes (e.g., ras and braf and egfr), including multiple sites on one or more genes.

Brief Description of the Drawings of the Invention

Fig. IA is a graph showing the melting curves generated for a synthetic ssDNA template corresponding to a portion of wild-type ras spanning codon 12, hybridized with five different probes, and visualized using a double-stranded DNA binding dye.

Fig. IB is a graph showing the melting curves generated for a synthetic ssDNA template corresponding to a portion of ras spanning codon 12 and having a G 12V mutation, hybridized with five different probes, and visualized using a double-stranded DNA binding dye.

Fig. 1C is a graph showing the melting curves generated for a synthetic ssDNA template corresponding to a portion of ras spanning codon 12 and having a G12C mutation, hybridized with five different probes, and visualized using a double-stranded DNA binding dye.

Fig. ID is a graph showing the melting curves generated for a synthetic ssDNA template corresponding to a portion of ras spanning codon 12 and having a G12D mutation, hybridized with five different probes, and visualized using a double-stranded DNA binding dye. Fig. IE is a graph showing the melting curves generated for a synthetic ssDNA template corresponding to a portion of ras spanning codon 12 and having a G12R mutation, hybridized with five different probes, and visualized using a double-stranded DNA binding dye.

Fig. 2 is a graph showing the melting curves generated for a synthetic ssDNA template corresponding to a portion of ras spanning codon 12 and having a G12R mutation, hybridized with five different probes and visualized using SYTO® 9 double- stranded DNA binding dye.

Fig. 3A is a graph showing the melting curve generated using a 208 bp amplification product from a tumor sample with a G12R mutation in the ras gene. Fig. 3B is a graph showing the melting curve generated using an 82 bp amplification product from a tumor sample with a G12R mutation in the ras gene.

Fig. 3C is a graph showing the melting curve generated using a 208 bp amplification product from a tumor sample with a G 12V mutation in the ras gene.

Fig. 3D is a graph showing the melting curve generated using an 82 bp amplification product from a tumor sample with a G 12V mutation in the ras gene.

Fig. 4A is a graph showing melting curves generated using double-stranded or single-stranded DNA templates.

Fig. 4B is a magnified view of the graph of Fig. 4A.

Fig. 5 A is a graph showing optimization of bead concentration in the method of the invention (the plot of the raw fluorescence is shown).

Fig. 5B shows the derivative plot of the data in Fig. 5 A.

Fig. 6A is a graph showing optimization of double-stranded nucleic acid binding dye in the method of the invention (the plot of raw fluorescence is shown). Fig. 6B shows the derivative plot of the data in Fig. 6A.

Fig. 7A shows the method of the invention using gel extraction after the amplification step.

Fig. 7B shows the method of the invention using spin column after the amplification step.

Figs. 8A and 8B show melting curve analysis of a tumor sample containing mutant ras where PNA was added only in the first amplification step (Fig. 8A) or in both amplification steps (Fig. 8B).

Figs. 8C and 8D show melting curve analysis of a tumor sample containing mutant ras where PNA was added only in the first amplification step (Fig. 8C) or in both amplification steps (Fig. 8D).

Figs. 8E and 8F show melting curve analysis of a tumor sample containing wild- type ras where PNA was added only in the first amplification step (Fig. 8E) or in both amplification steps (Fig. 8F). Figs. 9A-9E are graphs showing the results of the genotyping method of the invention in five different tumor samples, as compared to concurrent sequence analysis. Each figure represents a different patient sample (Fig. 9A: wild-type; Fig. 9B: G 12V mutant; Fig. 9C: G12C mutant; Fig. 9D: G12C mutant; Fig. 9E: G12R mutant).

Figs. 1OA and 1OB show the results of the genotyping method of the invention analyzing tumor samples bearing two different ras mutations at codon 12 (Fig. 1OA: sample 070035; Fig. 1OB: sample 070070).

Fig. 11 is a graph illustrating the use of the method of the invention to identify the presence of novel mutations by monitoring the shape of the melting curves.

Fig. 12 is a graph showing an analysis of the sensitivity of the method of the invention.

Figs. 13A-13F are graphs showing a comparison of the method of the invention using either two-step PCR (Figs. 13 A, 13C and 13E) or one-step PCR (Figs. 13B, 13D and 13F) in three different clinical samples (Figs. 13A/B, Figs. 13C/D, and Figs. 13E/F).

Figs. 14A-14D are graphs showing detection of codon 61 in ras and that increasing purine content in the PNA molecules increases the sensitivity of the genotyping method (Fig. 14A = PNA61B; Fig. 14B = PNA61C; Fig. 14C = PNA61D; Fig. 14D = PNA61F).

Figs. 15A-15C are graphs showing that increased length of oligonucleotide probes improves the detection of ras codon 61 mutations (Fig. 15A = 15bp probe; Fig. 15B = 18bp probe; Fig. 15C = 19bp probe).

Figs. 16A-16D are graphs showing the use of the genotyping method of the invention to genotype four different FFPE samples (Fig. 16A = wild-type sample; Fig. 16B = Q61H (CAT) mutant sample; Fig. 16C = Q61H (CAC) mutant sample; Fig. 16D = Q61L sample).

Fig. 17 is a graph showing the sensitivity of the method with respect to detection of a mutation at codon 61 of ras.

Fig. 18 is a graph showing the use of the genotyping method to determine genotype at codon 600 of the braf gene. Fig. 19 is a graph showing the sensitivity of the method with respect to detection of a mutation in braf.

Figs. 2OA and 2OB are graphs showing the use of the genotyping method to detect genotypes in deletion mutants of the gene encoding EGFR (Fig. 2OA = cell line HCC827; Fig. 20B = cell line H 1975). Fig. 21 is a graph showing the sensitivity of the method with respect to detection of a mutation in the gene encoding EGFR.

Figs. 22A and 22B are graphs showing the use of multiplexing to determine the genotype of a sample at codons 12 and 61 of ras (Fig. 22A = codon 12; Fig. 22B = codon 61). Figs. 23A and 23B are graphs showing the sensitivity of the multiplexing genotyping approach in two different cell lines (Fig. 23A = Pane 10.05 (G12D); Fig. 23B = SW948 (Q61L).

Figs. 24A-24D are graphs showing the use of the multiplexing genotyping method to determine the genotype for ras codon 12 (Fig. 24A), ras codon 61 (Fig. 24B), braf (Fig. 24C) and egfr (Fig. 24D) in the cell line Pane 10.05. The results indicate wild-type status for braf and k-ras codon 61 and mutant status (G 12D) for k-ras codon 12 as expected for this cell line.

Figs. 25A-25D are graphs showing the use of the multiplexing genotyping method to determine the genotype for ras codon 12 (Fig. 25A), ras codon 61 (Fig. 25B), braf (Fig. 25C) and egfr (Fig. 25D) in the cell line SW948. The results indicate wt status for braf Jems codon 12, and EGFR, and mutant status (Q61L) for k-ras codon 61 as expected for this cell line.

Figs. 26A-26D are graphs showing the use of the multiplexing genotyping method to determine the genotype for ras codon 12 (Fig. 26A), ras codon 61 (Fig. 26B), braf (Fig. 26C) and egfr (Fig. 26D) in the cell line Colo205. The results indicate wt status for k-ras codons 12, 61, and EGFR, and mutant status (V600E) for braf as expected for this cell line. Figs. 27A-27D are graphs showing the use of the multiplexing genotyping method to determine the genotype for ras codon 12 (Fig. 27A), ras codon 61 (Fig. 27B), braf (Fig. 27C) and egfr (Fig. 27D) in the cell line HCC827. The results indicate wt status for k-ras codons 12 and 61 and braf and mutant status (E746-A750 del.) for EGFR as expected for this cell line. Detailed Description of the Invention

The present invention generally relates to reagents, products, kits and methods for the amplification and/or detection of one or more target sequences in a gene, including, but not limited to, mutations in a gene or polymorphisms in a gene. While the invention is applicable to the detection and identification of virtually any target sequence in any gene, it is particularly useful for detecting rare or underrepresented mutations in a gene (e.g., mutations that occur at a low frequency in a given nucleic acid sample, such as when only a subset of DNA within the sample is mutated, and the remainder is of the wild-type species). The invention is also useful for detecting small or point mutations, including substitutions or deletions, and may also be employed for identifying chromosomal rearrangements that form new junctional DNA sequences.

Accordingly, the invention more particularly relates to a diagnostic method and products, as well as assay systems and/or kits for the amplification, detection and identification of one or more target sequences that occur at a selected site in a target polynucleotide (e.g., a gene). The method of the invention is typically used to detect and identify target sequences that are known to exist in a target polynucleotide at the time the method is performed (e.g., one or more known mutations in a gene), in contrast to a method such as sequencing, which may detect any mutation or mutations in a given polynucleotide, whether previously known or not. For example, in human the K-ras gene, there are currently 19 different mutations that are known to occur within codon 12 of the gene (the codon encoding the amino acid at position 12 of the protein sequence). Therefore, in this example, the target polynucleotide is K-ras or a portion thereof, the selected site is codon 12, and the target sequence is a nucleic acid sequence that contains one or more of the currently known 19 mutations that can occur at this site. Notwithstanding the foregoing discussion, the method of the invention can also detect mutations or variants that are not directly targeted by the design of the assay; while the assay will not detect the exact variant in this circumstance, it can identify that there is a variation other than those being tested for in the sample. A demonstration of this type of analysis is shown in the Examples. Accordingly, the method of the invention may also allow the user to identify rare, novel, or unexpected genotypes that exist within the same selected site as the target sequence.

In addition, primers, probes, and/or PNA described herein, as well as the use of these tools for amplification of and detection of sequences in genetic material, while being useful in the genotyping method of the present invention disclosed herein, also have utility in and applicability to a wide range of genotyping methods, including sequencing methods. Moreover, few genotyping kits and methods currently available provide appropriate reagents for the detection of certain rare mutations, such as those described at codon 61 of ras herein, or provide a set of reagents that can efficiently detect rare mutations or deletions at multiple different codons of ras, B-raf, or egfr within a mixture that includes abundant wild-type genetic material. Therefore, the reagents described herein have utility beyond the genotyping method of the invention. Therefore, the invention also relates to reagents including without limitation any of the primers, probes and/or PNA described herein, for use to amplify, detect and/or identify target sequences using any genotyping method that can utilize such reagents {e.g., any method that requires or can include amplification and/or identification of a mutant sequence polynucleotide), including without limitation any hybridization method, primer extension method, single strand conformation polymorphism method, pyrosequencing method, high resolution melting method, or sequencing method. With respect to the genotyping method of the invention, this method has a number of desirable features that make it particularly useful for genotyping patient samples in a clinical setting. First, the method incorporates steps that enable the detection of one or more low frequency target sequences in a nucleic acid sample that has a high frequency of non-target sequences. Specifically, the method includes the use of reagents that inhibit the amplification, and therefore the subsequent detection, of non-target sequences in the sample, thereby allowing the amplification and enhanced detection of low frequency target sequences. These reagents are also useful in other genotyping/sequencing methods as described herein. The subsequent steps of the genotyping method of the invention further enhance the ability to detect the target sequence, if present, by further minimizing the presence of non-target sequences through an isolation step, and by the use of a detection method that readily differentiates between target and non-target sequences. Interference of non-target sequences in a genotyping method can be a problem, as the target sequence may be missed altogether or even mis-typed in an overabundance of non-target nucleotides in the reaction that sequester reagents and may mask the presence of the target or inhibit its proper identification.

The genotyping method of the invention is also able to identify specific target sequences, rather than merely providing a yes/no indication of the presence of a sequence difference at a particular site. This is particularly important, for example, when a diagnostic or therapeutic method relies on the identification of a specific mutation or genotype in a patient sample (e.g., because the therapeutic method is designed to target that particular mutation).

Another advantage of the method of the invention is that it can be used in multiplex reactions to detect multiple different target sequences within the same sample, including detecting multiple genotypes at the same site in a gene, detecting target sequences at different sites within the same gene, and/or detecting target sequences in multiple genes in a genome {e.g., genes that may be associated by their products' function in a biological pathway or by their contribution to the same disease or condition). Because the method of the invention does not require multiple dyes or other labeling agents to detect differences in target sequences, and because the method differentiates target sequences from non-target sequences based on a specific characteristic (melting temperature) that is unique to the target sequence as compared to non-target sequences, large multiplexing strategies are feasible and straightforward using the method of the invention.

Additionally, the first steps of the method are conducted in a single reaction, as opposed to requiring multiple different reaction conditions to differentiate target sequences. This reduces the consumption of the nucleic acid sample (important when the sample is of limited quantity and/or low quality, which can occur when using genomic DNA obtained from clinical samples), and thereby reduces concerns with contamination from transfer steps, and provides more consistent and reproducible results. Indeed, certain steps of the method of the invention in which target sequences are amplified, as well as the primers and other nucleic acid binding moieties used in these steps, can also be used to amplify target sequences for other genotyping methods in which amplification of a target sequence is desired.

More particularly, multiple different polynucleotides can be amplified in a single reaction in the first steps of the method without increasing the amount of sample nucleic acid used, thus allowing for large genotyping efforts even when the sample is limited. In addition, because the method produces relatively short amplicons for which the likelihood of successful amplification is high, the method is readily adaptable to lower quality nucleic acid samples, where production of longer amplicons might be difficult. The conditions and exact procedures by which collection of patient samples occurs may vary from clinical site to clinical site, and some procedures may produce lower quantity DNA than others. This, combined with potential issues during the handling, transport and storage of the samples can result in patient samples that have such low quality and/or a limited quantity of DNA that a genotyping method may not be adequate to provide an accurate result, or to determine the genotype at all. For example, if a given genotyping method requires multiple initial reactions in which the DNA must be aliquoted among the reactions, the quantity of DNA may be insufficient to complete the test, resulting in a failed test, increased false negatives, and/or the inability of a patient to timely receive therapy because the sampling and diagnostic were insufficient to genotype the patient. However, the methods and reagents of the present invention can be used on patient samples of very low quantity and/or quality, and still provide a robust genotyping of the patient sample with respect to multiple target sequences, in both the genotyping method of the invention, and other genotyping methods.

The design of the method also eliminates concerns with competition or interference between the amplification reagents and those used to detect the target sequences, because the amplification step and the detection steps are carried out independently of one another. Such competition can be a problem in a method that relies on hybridization of reagents with nucleic acid sequences for both amplification and detection. Accordingly, the method is amenable to the use of a wider variety of detection reagents, amplification conditions, and detection conditions. The method of the present invention is also rapidly performed, being completed in just a few hours, and requires only standard laboratory equipment and reagents to perform. This is important in a clinical setting, where the ability to transfer the method from one laboratory to another and complete the method within a few hours enables the facile and rapid screening of more patient samples and a quicker time to diagnosis or prognosis, not to mention a lower cost basis for the assay. This translates to more rapid deployment of a particular therapeutic protocol for the patient.

The method of the invention can also be automated and thus can be used in a high throughput assay format. With respect to the multiplexing feature of the method, this enables the high throughput screening for multiple mutations or other genotypes, in multiple sites of a single gene, and/or in multiple genes in a patient sample. The method of the present invention can be used to rapidly detect, from a single patient sample (or multiple patient samples), not only specific mutations or variations in a gene or genes, but also collections of variations and mutations that may provide information about a particular biological pathway in a patient, or result from a particular therapeutic approach (e.g., escape mutations), and can inform the clinician about the overall health, proper diagnosis, prognosis, and optimum treatment pathway for the patient.

In addition, the method of the invention can be used to genotype pathogens that have infected a patient, such as viruses. By applying the method of the invention to RNA species, the method can also be used to detect nucleic acids that are not found by screening DNA (variations resulting from RNA editing), or can be used to "phenotype" a patient by screening mRNA, or to type the abundant species of rRNA or tRNA. Finally, the reagents provided herein expand the applicability of certain embodiments and aspects of the invention related to these reagents to other genotyping methods, including any genotyping method that requires or would benefit from an amplification step and including sequencing methods. Given the guidance provided herein, other applications of the invention will be clear. General Techniques and Definitions The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Methods of Enzymology, Vol. 194, Guthrie et al, eds., Cold Spring Harbor Laboratory Press (1990); Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly referred to herein as "Sambrook"); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000.

An "individual", "subject" or "patient" is a vertebrate, preferably a mammal, more preferably a human. An isolated "nucleic acid molecule" or "polynucleotide" is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation), its natural milieu being the genome or chromosome in which the nucleic acid molecule is found in nature. As such, "isolated" does not necessarily reflect the extent to which the nucleic acid molecule has been purified, but indicates that the molecule does not include an entire genome or an entire chromosome in which the nucleic acid molecule is found in nature. An isolated nucleic acid molecule can include a gene, and can be as small as an oligonucleotide. An isolated nucleic acid molecule that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes that are naturally found on the same chromosome. An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end of the sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., heterologous sequences). Isolated nucleic acid molecule can include DNA, RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA). Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides that comprises the nucleic acid molecule, the two phrases can be used interchangeably.

As used herein, the term "homologue" when used with reference to a polynucleotide or other nucleic acid binding agent {e.g., peptide nucleic acid) is used to refer to polynucleotide or other nucleic acid binding agent which differs from a naturally occurring polynucleotide or other nucleic acid binding agent (i.e., the "prototype" or "wild-type" polynucleotide) by minor modifications to the naturally occurring polynucleotide or other nucleic acid binding agent, but which maintains the basic structure of the naturally occurring form or has similar functional properties as the reference polynucleotide or other nucleic acid binding agent {e.g., similar binding properties). Such changes include, but are not limited to: changes in one or a few nucleotides, peptides, or other moieties, including deletions, insertions and/or substitutions. Derivatives of a polynucleotide or other nucleic acid binding agent may also be considered to be a homologue of a polynucleotide or other nucleic acid binding agent. For example, a nucleic acid binding agent such as a peptide nucleic acid that has similar functional properties as a polynucleotide (e.g., binds to the same nucleic acid sequence) can be considered a derivative or homologue of the polynucleotide. In one aspect, a homologue may have similar properties as the reference polynucleotide or other nucleic acid binding agent, such as a similar ability to bind to a target sequence. In another aspect, a homologue may have increased or decreased properties as the reference polynucleotide or other nucleic acid binding agent. Homologues can be produced using techniques known in the art including, but not limited to, modifications to the nucleic acid sequence for example, using classic or recombinant DNA techniques to effect random or targeted mutagenesis.

A homologue of a given polynucleotide or other nucleic acid binding agent may comprise, consist essentially of, or consist of, a sequence that is at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least about 99% identical (or any percent identity between 45% and 99%, in whole integer increments), to the sequence of the reference polynucleotide or other nucleic acid binding agent. In one embodiment, the homologue comprises, consists essentially of, or consists of, a sequence that is less than 100% identical, less than about 99% identical, less than about 98% identical, less than about 97% identical, less than about 96% identical, less than about 95% identical, and so on, in increments of 1%, to less than about 70% identical to the naturally occurring sequence of the reference polynucleotide or other nucleic acid binding agent.

Reference to an isolated protein or polypeptide in the present invention includes full-length proteins, fusion proteins, or any fragment, domain, conformational epitope, or homologue of such proteins. More specifically, an isolated protein, according to the present invention, is a protein (including a polypeptide or peptide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for example. As such, "isolated" does not reflect the extent to which the protein has been purified. Preferably, an isolated protein of the present invention is produced recombinantly. According to the present invention, the terms "modification" and "mutation" can be used interchangeably, particularly with regard to the modifications/mutations to the amino acid sequence of proteins or portions thereof (or nucleic acid sequences) described herein.

A "target polynucleotide" refers to any polynucleotide that is amplified in a method of the invention for detection of a sequence of interest (a "target sequence") within such polynucleotide. The term "target polynucleotide" may be used interchangeably with the term "template" when used to describe the starting material for an amplification reaction. The target polynucleotide can be any nucleotide species, including DNA, RNA, a DNA/RNA hybrid, or an RNA/RNA hybrid, and includes single- or double-stranded polynucleotides. A target polynucleotide can include an entire gene or any portion of a gene (or product of transcription thereof) that is of a sufficient length to be amplified (e.g., using polymerase chain reaction (PCR)) to produce an amplification product. A "non-target polynucleotide" refers to any polynucleotide that is not targeted

(designated, intended) for amplification using a method of the invention. Most typically, a non-target polynucleotide refers to a nucleic acid species that is highly related to the target polynucleotide, such as a variation of the same polynucleotide, but that does not contain the target sequence at a selected site in the polynucleotide. For example, a non-target polynucleotide can be a portion of a gene having a wild-type sequence at a selected site (the wild-type gene), where the target polynucleotide is a portion of the same gene, but has a mutation at the selected site (a mutated gene). As another example, a non-target polynucleotide can be one variant of a gene which contains a particular nucleic acid sequence at a selected site, and a target polynucleotide can be a different variant of the same gene that contains a different sequence (the target sequence) at the selected site. Accordingly, examples of non-target and target polynucleotides can include, but are not limited to, wild-type and mutated versions of the same gene, different mutated versions of the same gene, allelic variants of the same gene, splice variants of the same gene, or any polymorphic versions of essentially the same nucleic acid sequences. A "target sequence", as used herein, refers to any nucleic acid sequence that may occur within the target polynucleotide and that is targeted for detection (intended to be detected) using the method of the invention.

A "selected site" refers to the portion of, or site within, the target polynucleotide, where a given target sequence (e.g., a mutation, a variation, or the nucleotides defining a particular genotype) may occur. For example, a selected site can include, but is not limited to, a single nucleotide, a codon, or any sequence comprising two or more consecutive nucleotides that define a site of interest and where a variation or mutation or other genotype of interest can occur (but may or may not occur) in the target polynucleotide. The term "selected" infers that the site is predetermined, so that the appropriate method reagents (e.g., amplification primers, inhibitory agents, hybridization probes, etc.) can be designed and produced.

An "amplicon" as used herein refers to a polynucleotide formed as the product of natural (e.g., by gene duplication) or artificial (e.g., by PCR or ligase chain reaction) amplification events.

The term "primer" as used herein refers to an oligonucleotide that serves as a starting point for DNA replication by annealing to (hybridizing to) a nucleic acid template under appropriate conditions. Primers may be fully complementary to the portion of the nucleic acid template to which they anneal, or they may contain one or more non- complementary nucleotides.

A "hybridization probe" (also referred to as a "probe") is a nucleic acid binding agent, which can include, but is not limited to, an oligonucleotide, a peptide nucleic acid molecule, or any derivative or analog thereof, that is used to identify a target nucleic acid sequence by hybridizing to such target nucleic acid sequence under stringent hybridization conditions. A probe typically binds to its target because the probe has a structure or sequence that is complementary to at least a portion of the target sequence.

A "hybridized polynucleotide" (also referred to as a "hybrid") refers to a polynucleotide that is formed by the hybridization (base pairing, binding) between two single-stranded polynucleotides or between a single-stranded polynucleotide and another nucleic acid binding agent (e.g., a peptide nucleic acid), including without limitation a hybridization probe.

The "melting temperature" or "T_m" of any polynucleotide as used herein is defined as the temperature at which half of the polynucleotide strands are in the double-helical state and half are in the "random-coil" state. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule. A variety of methods can be used to predict or estimate T_m values, although it is noted that such calculations are imperfect and are not a substitute for empirically determined T_m values. The method of the invention calculates T_m based on the global maximum of the first derivative of the raw fluorescence data. Methods of the Invention

One embodiment of the present invention relates to a method of amplifying one or more target polynucleotides from a nucleic acid sample to produce at least one amplification product (amplicon), each amplification product containing a selected site of a target polynucleotide. In one aspect of this method of the invention, amplification of target polynucleotides having a non-target sequence at the selected site is inhibited, thereby enhancing amplification of target polynucleotides having a target sequence at the selected site. In another aspect, amplification of polynucleotides having a non-target sequence at the selected site is not inhibited. In the case where inhibition is utilized, such inhibition of the amplification of the non-target sequence {e.g., a wild-type sequence or non-targeted variant sequence) allows for the amplification of rare or underrepresented mutations in a nucleic acid sample, which may be otherwise difficult or impossible to detect in a background of non-target polynucleotide. The details for the procedure for amplification of a target polynucleotide are provided below.

The specific sequences and optional pairing of primers for the amplification method of the invention are described in detail below and in the Examples section. However, it is noted that one need not use the specific pairs of primers described herein for amplification of a target polynucleotide, since combination of a first primer that is described herein with a second primer described herein that is different than the primer pairing used in the Examples, or pairing of such first primer with another suitable second primer that is not specifically described herein {e.g., a second primer designed or generated outside of this invention) can be used to amplify a target polynucleotide. Accordingly, this embodiment of the invention encompasses the use of at least one primer described herein to amplify a target polynucleotide. The method may optionally use at least one PNA sequence described herein for inhibition of the amplification of non-target polynucleotides, but one may use any suitable PNA or other inhibitory nucleic acid binding moiety in an amplification method that includes at least one primer described herein. Similarly, one may use at least one PNA sequence described herein for the inhibition of the amplification of non-target polynucleotides in an amplification method that utilizes any suitable primers, including primers that are not specifically described herein, including primers available in the art or designed or generated outside of this invention.

In one aspect of the invention, a PNA is selected that binds to a sequence of a target polynucleotide which overlaps with at least a portion (is the same as at least a portion) of the sequence to which the primers used for amplification also bind. Such an example is provided herein. A non-limiting example of such a PNA and primer combination is shown in the use of the PNA of SEQ ID NO:26 to inhibit amplification of wild-type DNA in an amplification performed using primers SEQ ID NO:29 and SEQ ID NO:30.

This embodiment of the invention, which uses the specific primers and/or PNA sequences described herein, can be utilized to amplify one or more target polynucleotides for any suitable research, clinical, or diagnostic purpose, including without limitation, for any genotyping method that requires or would benefit from a step of amplification of a target polynucleotide, either as a part of the genotyping method or prior to employing the genotyping method. Such genotyping methods include, but are not limited to, the genotyping method of the present invention, sequencing methods including bi-directional sequencing, any genotyping methods that include hybridization of probes or other nucleic acid binding moieties to a target sequence, or any genotyping methods that include extension, sequence detection or sequence determination of an amplified target sequence. Various genotyping methods include, but are not limited to, hybridization methods, primer extension methods, single strand conformation polymorphism methods, pyrosequencing methods, high resolution melting methods, and sequencing methods.

In this embodiment of the invention, the target polynucleotide is from a gene selected from ras, b-raf, or egfr {i.e., genes encoding Ras, B-Raf, or EGFR, respectively). As discussed above, a target polynucleotide is any portion of the gene (or product of transcription thereof) that is of a sufficient length to be amplified {e.g., using polymerase chain reaction (PCR), or reverse transcriptase PCR (RT-PCR) in the case of the transcription product) to produce an amplification product.

In one aspect of this embodiment of the invention, when the target polynucleotide is from ras, the selected site is a nucleic acid sequence that is within exon 2 of ras, which can include a sequence that comprises codon 12 of ras, and/or a nucleic acid sequence that includes codon 13 of ras. In one aspect of this embodiment of the invention, when the target polynucleotide is from ras, the selected site is a nucleic acid sequence that is within codon 13 of ras, which can include a sequence that comprises codon 59 of ras, a nucleic acid sequence that includes codon 61 of ras, a nucleic acid sequence that includes codon 76 of ras, and/or a nucleic acid sequence that includes codon 146 of ras. The primers described herein for amplification of a nucleic acid sequence within exon 2 of ras may include more than one codon, e.g., a primer for amplification of a nucleic acid sequence that includes codon 12 of ras can also be used to amplify a nucleic acid sequence that includes codon 13 of ras, as well as other codons within the amplicon produced using the codon 12 primers (described below). Similarly, the primers described herein for amplification of a nucleic acid sequence from exon 3 of ras that includes codon 61 of ras can also be used to amplify a nucleic acid sequence that includes codon 59 or codon 76 of ras, as well as other codons within the amplicon produced using the codon 61 primers (described below).

In one aspect, this method of the invention includes an oligonucleotide primer or homologue thereof, that hybridizes to, and/or is used for amplification of, sequences from exon 2 of ras, and which include codon 12 of ras. These primers are selected from a primer having a sequence comprising or consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, or SEQ ID NO:58. Homologues of these sequences are expressly encompassed by the invention. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above-identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, SEQ ID NO: 7 with any of SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:11; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:53 and SEQ ID NO:54, or SEQ ID NO:57 and SEQ ID NO:58. Any other combination of forward and reverse primers selected from the above primers is also encompassed by the invention.

PNA useful for the inhibition of the amplification of a portion of wild-type ras comprising codon 12 includes a PNA that comprises or consists of the sequence of SEQ ID NO: 17, or a homologue thereof. As discussed above, the PNA of the invention can be used to inhibit the amplification of wild-type ras in conjunction with the use of any amplification primers, including primers designed or developed inside or outside of this invention. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type ras in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds. In one aspect, this method of the invention includes an oligonucleotide primer or homologue thereof that hybridizes to, and/or is used for amplification of, sequences from exon 3 of ras and which include codon 61 of ras. These primers are selected from a primer having a sequence comprising or consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:59, or SEQ ID NO:60. Homologues of these sequences are expressly encompassed by the invention. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above-identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, the combination of SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:27 and SEQ ID NO:30, SEQ ID NO:27 and SEQ ID NO:48, SEQ ID NO:27 and SEQ ID NO:56, SEQ ID NO:27 and SEQ ID NO:60, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:29 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:48, SEQ ID NO:29 and SEQ ID NO:56, SEQ ID NO:29 and SEQ ID NO:60, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:47 and SEQ ID NO:56, SEQ ID NO:47 and SEQ ID NO:60, SEQ ID NO:47 and SEQ ID NO:28, SEQ ID NO:47 and SEQ ID NO:30, SEQ ID NO:55 and SEQ ID NO:56, SEQ ID NO:55 and SEQ ID NO:48, SEQ ID NO:55 and SEQ ID NO:60, SEQ ID NO:55 and SEQ ID NO:28, SEQ ID NO:55 and SEQ ID NO:30, SEQ ID NO:59 and SEQ ID NO:60, SEQ ID NO:59 and SEQ ID NO:48, SEQ ID NO:59 and SEQ ID NO:56, SEQ ID NO:59 and SEQ ID NO:28, or SEQ ID NO:59 and SEQ ID NO:30.

PNA useful for the inhibition of the amplification of a portion of wild-type ras comprising exon 3, including codon 61 includes a PNA that comprises or consists of the sequence of: SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, or a homologue thereof. As discussed above, the PNA of the invention can be used to inhibit the amplification of wild-type ras in conjunction with the use of any amplification primers, including primers designed or developed inside or outside of this invention. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type ras in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds. For example, in one aspect, the PNA of SEQ ID NO:26 inhibits amplification of wild-type ras in an amplification reaction in which SEQ ID NO:29 and/or SEQ ID NO:30 are used. In another aspect of the invention, when the target polynucleotide is from braf, the selected site is a nucleic acid sequence that includes codon 600 of braf. The primers described herein for amplification of a nucleic acid sequence that includes codon 600 of braf can also be used to amplify a nucleic acid sequence that includes other codons within the amplicon produced using the codon 600 primers (described below).

In one aspect, this method of the invention includes an oligonucleotide primer or homologue thereof that hybridizes to, and/or is used for amplification of, sequences that include codon 600 of brαf These primers are selected from a primer having a sequence comprising or consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:49 or SEQ ID NO:50. Homologues of these sequences are expressly encompassed by the invention. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above-identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, SEQ ID NO:36 and SEQ ID NO:37, or SEQ ID NO:49 and SEQ ID NO:50. Any other combination of forward and reverse primers selected from the above primers is also encompassed by the invention.

PNA useful for the inhibition of the amplification of a portion of wild-type brαf comprising codon 600 includes a PNA that comprises or consists of the sequence of SEQ ID NO:38 or a homologue thereof. As discussed above, the PNA of the invention can be used to inhibit the amplification of wild-type brαf in conjunction with the use of any amplification primers, including primers designed or developed inside or outside of this invention. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type brαf in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds.

In another aspect of the invention, when the target polynucleotide is from the gene encoding EGFR, the selected site is a nucleic acid sequence that includes a portion of exon 19 of the gene encoding EGFR. The primers described herein for amplification of a nucleic acid sequence that includes codon exon 19 of egfr can also be used to amplify a nucleic acid sequence that includes other codons within the amplicon produced using these primers (described below).

In one aspect, this method of the invention includes an oligonucleotide primer or homologue thereof that hybridizes to, and/or is used for amplification of, sequences from exon 19 of the gene encoding EGFR. These primers are selected from a primer having a sequence comprising or consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:51 or SEQ ID NO:52. Homologues of these sequences are expressly encompassed by the invention. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above -identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, SEQ ID NO:41 and SEQ ID NO:42 or SEQ ID NO:51 and SEQ ID NO:52. Any other combination of forward and reverse primers selected from the above primers is also encompassed by the invention.

PNA useful for the inhibition of the amplification of a portion of wild-type gene encoding EGFR includes a PNA that comprises or consists of the sequence of SEQ ID NO:43 or a homologue thereof. As discussed above, the PNA of the invention can be used to inhibit the amplification of wild-type EGFR gene in conjunction with the use of any amplification primers, including primers designed or developed inside or outside of this invention. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type egfr in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds.

Yet another embodiment of the present invention relates to the use of hybridization probes (e.g., oligonucleotide probes) for the detection of target sequences in a nucleic acid sample, wherein the target sequences are within a target polynucleotide from a gene selected from ras, braf, or egfr. Such probes can be used in any method that includes a step of using a hybridization probe to detect the presence of a specific sequence in a polynucleotide, including but not limited to, the genotyping method described herein, and any other genotyping method in which an oligonucleotide probe is employed. The use of probes to hybridize to a sequence or detect a sequence is described in detail below with respect to the genotyping method of the invention, but the basic steps of hybridizing a probe to a target sequence are encompassed for general use as part of this embodiment of the invention. The use of each of the probes encompassed by the invention is also described in the Examples, as are the sequences of the probes.

Probes useful for the hybridization to and detection of a target sequence within exon 2 of ras, which may include wild-type and/or mutated ras sequences, include probes with a sequence comprising: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, or a homologue thereof.

Probes useful for the hybridization to and detection of a target sequence within exon 3 of ras, which may include wild-type and/or mutated ras sequences, include probes with a sequence comprising: SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35, or a homologue thereof.

Probes useful for the hybridization to and detection of a target sequence within braf, which may include wild-type and/or mutated braf sequences, include probes with a sequence comprising: SEQ ID NO:39 or SEQ ID NO:40, or a homologue thereof.

Probes useful for the hybridization to and detection of a target sequence within exon 19 of the egfr gene, which may include wild-type and/or mutated sequences, include probes with a sequence comprising: SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46, or a homologue thereof.

Another embodiment of the present invention relates to a genotyping method for detecting target sequences in a nucleic acid sample. The method includes the steps of: (a) amplifying one or more target polynucleotides from a nucleic acid sample to produce at least one amplification product, each amplification product containing a selected site of a target polynucleotide for detection of target sequences;

(b) isolating single-stranded polynucleotides from the amplification product;

(c) contacting the single stranded polynucleotides with a hybridization probe and with a label that detects hybridization of a single-stranded polynucleotide and the hybridization probe, under conditions sufficient to cause the single-stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide;

(d) detecting the melting temperature (T_m) of the hybridized polynucleotide; and (e) detecting target sequences at the selected site of the target polynucleotides by detecting perfectly hybridized polynucleotides, wherein perfectly hybridized polynucleotides have a higher T_m than hybridized polynucleotides formed with a probe having a nucleic acid sequence that is different from the single-stranded polynucleotide at the selected site by at least one nucleotide. In the method of the invention, during step (a), amplification of target polynucleotides having a non-target sequence at the selected site is inhibited, thereby enhancing amplification of target polynucleotides having a target sequence at the selected site. Inhibition of the amplification of the non-target sequence (e.g., a wild-type sequence or non-targeted variant sequence), in combination with the other steps of the method of the invention, allows for the detection of rare or underrepresented mutations in a nucleic acid sample, which may be otherwise difficult or impossible to detect in a background of non- target polynucleotides. For example, in one aspect of the invention, patient DNA is isolated from paraffin embedded tissue sections of the patient's tumor. The presence of normal stromal cells and large numbers of infiltrating lymphocytes in addition to tumor cells in this sample contributes to a significant wild-type genomic background. In addition, most mutations in tumor DNA are heterozygous, leaving 50% of the tumor genetic material as wild-type. Therefore, it is critical that the wild-type signal be suppressed sufficiently during the amplification, so that a detectable level of the mutant signal is obtained.

In step (c) of the method of the invention, the hybridization probe is configured to hybridize to a nucleic acid sequence spanning the selected site of a target polynucleotide. The hybridization probe has a nucleic acid sequence (or other structure, in the case of probes that are not nucleic acids per se, or that are derivatives) at the selected site corresponding to either (i) a target sequence, or (ii) a non-target sequence. This allows the detection of both target sequences and, as a control, non-target sequences in the patient population, and provides a basis for comparison during the detection process. In one aspect of the method (discussed in detail below), by using a plurality of different hybridization probes spanning the range of possible target sequences at the selected site, the method can identify the specific target sequence or lack thereof, because a hybridized polynucleotide formed with a probe that is fully complementary to the single-stranded polynucleotide (a "matched" sequence, or a "perfectly hybridized polynucleotide") will have a higher melting temperature (measured by determining T_m) than a hybridized polynucleotide formed with a probe having a nucleic acid sequence that is different from the single-stranded polynucleotide at the selected site by one or more nucleotides (a "non- matched" sequence, or "imperfectly hybridized polynucleotide"). Therefore, one can identify the probe that is a "match" for the target polynucleotide at the selected site, if any, and accordingly, identify the target sequence at the selected site. Amplification Step

The first step in the method of the invention is an amplification step. In addition, the details regarding amplification below may also be applied to the method described above for amplifying a target polynucleotide using any of the primers and PNA described herein for any other method that requires or benefits from polynucleotide amplification. Specifically, one or more target polynucleotides are amplified from a nucleic acid sample to produce at least one amplification product. The amplification step is designed (e.g., by the predetermined selection of the polynucleotide to be amplified) to specifically produce an amplification product that contains a selected site of a target polynucleotide for detection of target sequences.

The first step of this method of the present invention includes amplifying at least one target polynucleotide from a nucleic acid sample (also referred to herein as a "test sample", or simply "sample") to produce at least one amplification product. A nucleic acid sample is any sample of genetic material (DNA or RNA or derivatives and hybrids thereof, including genomic DNA, cDNA, mRNA, tRNA, rRNA, DNA/RNA hybrids, RNA/RNA hybrids, etc.) that can be obtained from a patient or other source (cell lines, viruses, pathogens, synthetic sources, laboratory processes for creation or manipulation of nucleic acids, etc.). In one aspect of the invention, a suitable nucleic acid sample is genomic DNA obtained from cells of a patient. In another aspect, a suitable nucleic acid sample is RNA or DNA obtained from virus that has infected a patient. Suitable methods for obtaining a nucleic acid sample are known to a person of skill in the art, and typically begin with the collection or retrieval of a source of the nucleic acids from a patient. A patient sample can include any cells, or any bodily fluid, solid, tissue or organ from a patient that may contain cells of interest (e.g., tumor cells), or nucleic acids from such cells. For example, cells and tissues can be obtained by scraping of a tissue, biopsy (cutting, slicing, punch, needle biopsy, laser capture microscopy), processing of a tissue sample to release individual cells, or other isolation from an initial sample. In one aspect, patient nucleic acids are isolated or extracted from cells obtained using laser capture microscopy (LSM; see, e.g., Emmert-Buck et al, 1996, Science 274 (5289):998-1001). In another aspect, patient nucleic acids are isolated or extracted from cells obtained by a fresh fine needle aspirate sample. In yet another aspect, patient nucleic acids are isolated or extracted from needle scraped tumor cells. In one aspect of the invention, patient DNA is isolated or extracted from paraffin embedded tissue sections of a patient's tumor. In one aspect of the invention, viral nucleic acids may be isolated from an infected patient, such as by collecting the infected patient's blood or by obtaining biopsies, such as a fine needle aspirate. In another aspect of the invention, fetal nucleic acids may be isolated from a fetus by, for example, isolation of fetal DNA from maternal or fetal blood, or by a fine needle aspirate from the fetus.

After obtaining a sample of cells, tissue, fluids or solids from a patient, the nucleic acids are typically isolated or extracted from the cells prior to the amplification step. Methods for extracting nucleic acids from cells or tissues are well known in the art. For example, cells are lysed using a lysis buffer and proteinase solution and are typically incubated overnight, or until lysis is complete. If DNA is to be extracted, RNAse is also added to destroy RNA species, and the sample is washed and extracted DNA is collected. The nucleic acid extraction can be performed by the user of the method of the invention, or it can be performed by a different laboratory, including by the clinical laboratory where the sample is initially isolated. In one aspect of the invention, a patient sample is collected by a clinician, and the clinical laboratory associated with the clinician (or the clinician himself/herself) places the sample into a suitable lysis buffer before transferring/sending the sample to a laboratory for genotyping using the method of the invention. In this manner, the sample will arrive at the genotyping laboratory ready for final extraction, which significantly expedites the performance of the method by the laboratory performing the genotyping. In cases where RNA is the nucleic acid species to be genotyped, the samples will be DNAse treated and then subjected to reverse transcription-PCR (RT- PCR). The latter is best conducted on fresh cells/samples rather than paraffin embedded or otherwise fixed samples, because RNA integrity is superior for fresh tissues. The amount of nucleic acid sample required to perform the present method is an amount sufficient to allow for the amplification of one or more target polynucleotides from the sample, using one or more rounds of amplification (e.g., one or more PCR reactions). The present inventors have found that the method of the invention is useful for genotyping samples that contain low amounts of the nucleic acids or low quality nucleic acids, and can be used when the target polynucleotide, as a percentage of the total nucleic acids in the sample, or as a percentage of the total target and non-target polynucleotides in the sample (e.g., mutant and wild-type species of the same gene), is very low. In general, it is preferable to begin the amplification step with a nucleic acid sample that is at a concentration of from about 10 ng/μl to about lμg/μl, and more preferably, about 50 ng/μl to about 500 ng/μl. In one aspect, a suitable total amount of nucleic acid sample for use in the present method is between about 10 ng nucleic acids and about 1 mg for most samples (purified plasmid DNA or synthetic templates could be provided in a lower amount), and in one aspect, is between about 50 ng and about 500 ng, and in one aspect, is between about 100 ng and about 300 ng, and in one aspect, is about 200 ng, including any amount of nucleic acids between 10 ng and 1 mg, in whole integer increments (10 ng, 11 ng, 12 ng...998 ng, 999 ng, 1 mg). The method of the present invention is highly sensitive, detecting as little as 0.05% mutant polynucleotide in a sample, or detecting as little as 0.25ng mutant cells in a sample, and so very little starting material is required to detect a mutant signal.

Once a suitable nucleic acid sample is available (a "suitable" sample being any nucleic acid sample that is sufficiently extracted or isolated from its original source that it can be used in an amplification process), the target polynucleotide(s) is amplified from the sample to produce an amplification product. According to the present invention, "amplifying" refers to an increase in the number of copies of a target polynucleotide, which is typically an exponential increase. An "amplification product" is the end product of the target polynucleotide that has been increased in copy number as a result of amplifying it from the nucleic acid sample. The term "amplification product" can generally be used interchangeably with the term "amplicon", particularly when PCR is used.

Amplifying nucleic acids can be performed by any suitable method, which can include, but is not limited to, polymerase chain reaction (PCR; see, e.g., U.S. Patent Nos. 4,683,202; 4,683,195; and 4,965,188), ligase chain reaction (LCR; see, e.g., Wiedmann et al, 1994, PCR Methods and Applications, 3(4):S51-64), Q-Beta (Qβ) RNA replicase (see, e.g., Lizardi, et al., 1988, Biotechnology 6, 1197), and RNA transcription-based (TAS) amplification systems (see, e.g., Fahy et al., 1991, PCR Methods and Applications 1 :25- 33), strand displacement amplification (SDA; see, e.g., Walker et al., 1994, Nucleic Acids Research, 22(13): 2670-2677); nucleic acid sequence-based amplification (NASBA; see, e.g., Deiman et al., 2002, MoI Biotechnol. 20(2): 163-79); cascade rolling circle amplification (CRCA; see, e.g., Demidov, 2002, Expert Review of Molecular Diagnostics, 2(6): 542-548); isothermal and chimeric primer initiated amplification of nucleic acids (ICAN; see, e.g., Mukai et al., 2007, J. Biochem. 142(2):273-281); and the like. When RNA species are amplified, the amplification may include a reverse transcription step. Other nucleic acid amplification methods are known in the art and will be developed, and although PCR is frequently mentioned as one amplification method, it should be understood that the invention includes variations on PCR and other alternative amplification methods. In one aspect of the invention, the amplification step (amplifying step) is performed using PCR. While this method is well-known in the art, briefly, a DNA polymerase is used to amplify a target polynucleotide by in vitro enzymatic replication. Oligonucleotide primers (primer pairs) are designed to hybridize to opposite strands of the targeted nucleotide sequence, flanking the ends of the target polynucleotide sequence to be amplified. As PCR progresses through repeated cycles of primer annealing steps and extension steps, the DNA generated by these steps is itself used as a template for replication. The DNA template is thereby exponentially amplified through this chain of events. With PCR it is possible to amplify a single or few copies of a target polynucleotide across several orders of magnitude, generating millions or more copies of the target polynucleotide.

As discussed above, the term "primer" as used herein refers to an oligonucleotide that serves as a starting point for DNA replication. The primer anneals to (hybridizes to) a template (the target polynucleotide sequence) and synthesis of DNA by primer extension is initiated under appropriate conditions {e.g., the presence of a DNA polymerase, nucleotide triphosphates and suitable buffers, all used at appropriate temperatures). The size of the primer can be dependent on nucleic acid composition and percent homology or identity between the primer and the template to be amplified, as well as upon hybridization conditions per se (e.g., temperature, salt concentration, etc.). The size of a nucleic acid molecule that is used as an oligonucleotide primer is typically at least about 15 nucleotides and up to about 50 nucleotides in length, and explicitly includes any length in between, in whole integer increments (i.e., 15, 16, 17...20...30...40...48, 49, 50). A primer having a GC-rich nucleotide content may be shorter than a primer having an AT-rich content. Primers may be fully complementary to the portion of the target nucleotide to which they anneal, or they may contain one or more non-complementary nucleotides or even be included in mixtures of degenerate oligonucleotides, as long as the primer contains a sufficient number of correctly placed complementary nucleotides that the amplification of the target polynucleotide will occur. It is preferable for the method of the invention that primers be designed and/or selected that will specifically amplify the target polynucleotide and not non-target polynucleotides. Examples of primers that can be used to amplify portions of a ras gene, using one or two rounds of PCR, or to amplify portions of a braf gene or a gene encoding EGFR, are described herein, but the genotyping method of the invention is not limited to the use of these primers. It is also preferable to design or select primers for use in the present method that have a similar melting temperature to one another, so that the annealing reaction occurs for both primers in the reaction substantially simultaneously and within a common set of thermal cycling conditions. In one aspect, the primers are designed to carry either a G or C nucleotide at the 3 ' end. Furthermore, by designing primers that are longer than typical primers (e.g., greater than 20 nucleotides), one can increase the specificity of the primer for the complementary strand of target polynucleotide, which improves the amplification of the target polynucleotide and reduces non-specific amplification. In addition, it is preferable in the method of the invention to use primers with a T_m that is sufficiently different from the T_m of any nucleic acid binding moiety that is used to inhibit amplification of a non-target polynucleotide (e.g., a PNA molecule), so that the annealing of the primers to the target polynucleotide template does not occur at the same temperature as the binding of the nucleic acid moiety to the target polynucleotide. For example, it is desirable to design primers with a Tm that is at least about 5°C, 6°C, TC, 8°C, 9°C, 10⁰C or more lower than the Tm of the inhibitor molecule. In addition, separating the T_ms for these components will decrease the possibility that the inhibitor molecule will bind to mismatched sequences and inhibit their amplification.

To produce an amplification product in the method of the invention, one or more amplification steps should be performed to generate a sufficient amount of the desired amplification product. Generally, when PCR is used to amplify the target polynucleotide, the method of the invention can be performed using either one or two rounds of PCR. In one aspect, the amplification step is performed using only one round of PCR, which shortens the total time needed to perform the method of the invention, and which is therefore preferred. In another aspect, two PCR reactions may be performed in order to produce an amplification product. Such a reaction is typically performed using nested primers, wherein a first PCR is performed using external (outer) primers that amplify a particular, larger nucleic acid sequence, followed by a second PCR performed using internal (inner) primers that amplify a second, smaller sequence internal to that amplified by the first round of PCR. The concept of nested PCR is well known in the art. Two amplification steps, such as two PCR steps, may be used, for example, when the quantity of the nucleic acid sample to be used in the amplification reaction is limited or when the quality of the DNA in the sample is low, and/or when producing larger amplicons (e.g., greater than 200 bp). However, the present invention can be performed even on low quality or a limited quantity of DNA using one PCR amplification step, and is not limited to the use of nested PCR.

In one aspect of the method of the invention, the amplification step is designed to amplify more than one amplification product. For example, by the selection and design of various pairs of primers and inhibitory molecules, as well as suitable common amplification conditions, one can amplify two or more different selected sites from the same gene {e.g., a sequence including codon 12 of ras and a second sequence including codon 61 of ras) and/or selected sites from two or more different genes {e.g., a selected site from ras, such as any of those described herein, and a selected site from the gene encoding BRAF, such as a site including codon 600). In the case where more than one target polynucleotide is amplified, inhibition of non-target sequences can be accomplished by the inclusion of an appropriate inhibitory molecule {e.g., PNA) for each target polynucleotide. By amplifying two or more different amplification products in the same amplification reaction, the multiplexing ability of the invention is significantly extended. In some circumstances, a nucleic acid sample may be available in a limited quantity, or the quality may be poor. In such cases, the ability to screen for multiple genotypes from a single amplification product is highly valuable. Moreover, screening for multiple genotypes in a single assay allows for complex genotyping and design of the method to analyze not only particular genetic variations of interest, but also patterns in genetic variations that may be informative at the level of a biological pathway, or at the level of a disease or condition. In the design of these multiplexing amplification reactions, one should optimize the primer sequence design and amplification conditions to maximize the amplification of all target polynucleotides of interest, and to reduce competition among primers and/or any nucleic acid binding moieties used to inhibit amplification of non- target sequences.

In one aspect of the method, at least one of the primers in each pair of primers used in the amplification step (a pair of primers represents the minimum two primers required to produce an amplification product) is labeled with a selection moiety. Addition of a selection moiety is one way to enable the isolation of single-stranded polynucleotides from the amplification product(s) during the next step of the genotyping method (discussed below). According to the invention, a selection moiety can include any moiety (molecule or compound) that can be attached (linked) to or incorporated into a primer and then used to select the polynucleotide strand that was amplified by extension of such primer. The selection moiety should only be attached to or incorporated into one of the primers in a pair, so that the two strands of the amplification product can be separated from one another. Either strand of the amplification product can be labeled with the selection moiety, at the discretion of the user of the method; in one aspect, the sense strand of the amplification product is labeled; in another aspect, the anti-sense strand of the amplification product is labeled. Being able to select the strand for later stages of the method of the invention has the advantage of allowing for optimized probe design (i.e., a strand/probe combination can be selected that provides the best result in later steps of the method). In either case, the probes for the later hybridization step are designed to bind to the strand that has been amplified using a selectable primer and then isolated. In one aspect of the invention, the selection moiety is biotin, which can be later selected by binding of the biotin to streptavidin. In one aspect, two, three, or multiple biotin moieties can be incorporated into the primer, which will further increase the ability to isolate a single-stranded polynucleotide in the next step of the method. In this aspect, selection moieties should be selected so that they do not negatively impact the T_m of the primers or resulting amplicons used in later steps. This principle can be applied to other selection moieties as well. Other suitable selection moieties include any moiety or molecule that will interact with another moiety or molecule such as, but not limited to, digoxygenin, any partner of any receptor- ligand pair, an antibody or fragment thereof, FITC, and the like. A selection moiety used in this step should be chosen that will not interfere with the amplification process, or any of the subsequent steps of the method, including the probe hybridization step. If more than one round of amplification is used in the amplification step (e.g., two rounds of PCR), the selection moiety need only be incorporated into the second, or last, round of amplification. During the amplification step, it is also a requirement of the method of the invention that the amplification of one or more non-target polynucleotides be inhibited (i.e., reduced, decreased, which can include, but does not require, elimination of non-target polynucleotides). As a result, competition for the PCR reagents by non-target polynucleotides is reduced and the PCR reagents will be made available to the target polynucleotides (those containing a target sequence), thereby enhancing the amplification of target polynucleotides from the nucleic acid sample. This step is critical if the target sequence to be detected is a rare or underrepresented (those that are present in a small percentage of total cells within a target tissue) sequence in the nucleic acid sample. For example, when detecting mutations in the ras gene from a patient tumor sample, the amount of DNA encoding wild-type Ras (Ras having no mutations at the selected site) may be substantially greater than the amount of DNA in the sample that encodes mutated Ras, for example, as a result of non-cancerous cells that surround and are associated with the tumor tissue sample. Accordingly, it is necessary to inhibit amplification of the wild- type ras DNA in order to properly genotype mutant ras DNA in the sample, and this principle applies to any target polynucleotide that is rare or underrepresented. If more than one round of amplification is used in the amplification step {e.g., two rounds of PCR), the inhibition of the non-target polynucleotide can take place during one or all of the amplification rounds. In one aspect, the inhibition of the non-target polynucleotide occurs during the first amplification step and optionally occurs during subsequent amplification steps. In one aspect, the inhibition of the non-target polynucleotide occurs during the first amplification step and does not occur during subsequent amplification steps. The inventors have found that inhibition of the amplification of non-target polynucleotides during the first round of amplification, if there is more than one round, is typically sufficient for the method of the invention.

The step of inhibiting amplification of a non-target polynucleotide can be performed using any suitable method for inhibition of amplification. For example, inhibition methods include, but are not limited to, clamping of the non-target sequence in a non-target polynucleotide by the use of a nucleic acid binding moiety, restriction endonuclease-mediated selective polymerase chain reaction, stop primer technology or ARMS technology. In one aspect, the step of inhibiting is performed in the presence of a nucleic acid binding moiety (a molecule or compound that is capable of binding to a nucleic acid molecule) that binds to a non-target sequence at the selected site and thereby inhibits the amplification of a target polynucleotide having the non-target sequence at the selected site. Such nucleic acid binding moieties include, but are not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino oligonucleotides, RNA (and modified RNA), and HyNARNA. As discussed above, the T_m of the nucleic acid binding moiety should be designed or selected to be high enough to avoid/reduce non-specific, mismatch binding with the target sequences, and also to provide enough separation from the T_m of any primers used during the amplification step that competition between the binding moiety and primers is minimized.

Peptide nucleic acid (PNA) is an artificially synthesized polymer similar to DNA or RNA and is used in biological research and medical treatments. PNA is not known to occur naturally. Briefly, DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine or lysine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. PNAs are depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the right (see, e.g., Egholm et al., 1993, Nature 365:566-568; and Paulasova and Pellestor, 2004, Annales de Gέnέtique 47:349-358). Since the backbone of PNA contains no charged phosphate groups, the binding between PNA/DNA strands is stronger than between DNA/DNA strands due to the lack of electrostatic repulsion. Therefore, the T_m (melting temperature) of a PNA/DNA hybrid may be several degrees higher than the T_m of a DNA/DNA hybrid. Examples of PNA used to inhibit amplification of a non-target sequence are provided in the Examples section, and may be used in any of the methods of the invention described herein. Locked nucleic acid (LNA) is a modified RNA species, where the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' and 4' carbons. The bridge "locks" the ribose in the 3'-endo structural conformation, which is often found in the A-form of DNA or RNA. These nucleotides can then be mixed into oligonucleotides {e.g., a probe that would bind to a non-target sequence) to enhance base stacking and backbone pre-organization, thereby increasing the thermal stability (measured by melting temperature) of the oligonucleotides (see, e.g., Kaur et al., 2006, Biochemistry 45 (23):7347-55).

Morpholino oligonucleotides are synthetic molecules which are the product of a redesign of natural nucleic acid structure (see, e.g., Summerton and Weller, 1997, Antisense & Nucleic Acid Drug Development 7:187-95). The morpholino oligonucleotides are typically about 25 bases long, and bind to complementary sequences of RNA by standard nucleic acid base-pairing. Morpholinos have standard nucleic acid bases like DNA, but the bases are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates (Summerton and Weller, ibid.). Accordingly, these oligonucleotides bind to a sequence (e.g., a non-target sequence in a non-target polynucleotide) and block the binding or access of other molecules to the sequence.

Other intercalating nucleic acids (e.g., HyNA® nucleic acids and INA®, both manufactured by PentaBase of Denmark) are other examples of nucleic acid analogues that have high-affinity binding to their targets (e.g., complementary DNA targets, which in the context of the invention would typically be non-target sequences in non-target polynucleotides). Intercalation occurs when ligands of an appropriate size and chemical nature fit themselves in between base pairs of DNA.

Other modified RNAs may also be used as a nucleic acid binding moiety to inhibit amplification of a non-target polynucleotide in the invention, particularly if these RNA types discriminate well between matched and unmatched sequences, such that they are useful . Antisense RNAs can also be used, as can antisense DNA sequences. In restriction endonuclease-mediated selective PCR, amplification by PCR is conducted under conditions in which a restriction endonuclease site is incorporated into a non-target sequence, followed by simultaneous or subsequent digestion with a restriction endonuclease to remove or inhibit the non-target amplicon. This method, and variations of the method, are described in the literature (see, e.g., Ward et al., 1998, American Journal of Pathology, Vol. 153, No. 2).

In stop-primer technology, synthetic oligonucleotides used in conjunction with a PCR process create incomplete complementary strands of certain non-target sequences during amplification, which stops the subsequent synthesis of those strands (see, e.g., U.S. Patent Publication No. 2007/0082343). Another useful technology for inhibition of the amplification of non-target sequences may include allele specific amplification (ARMS™), which in this invention would block amplification of non-target sequences through the design of primers that terminate at the selected site (e.g., codon 12 of ras), but are matched to the non-target sequence at the 3' end. The polymerase is therefore not able to extend the template and amplification of the non-target sequence does not occur.

The amplification product produced during the amplification step of the method of the invention should be designed to be of a size that is sufficient to allow for the binding of the primers and the nucleic acid binding moiety (if this method of inhibition is used) during the amplification step, and with respect to the genotyping method of the invention, should also be of a size that, when hybridized with a hybridization probe during later steps of the method, results in a T_m for the hybrid that optimizes the ability to distinguish matches from mismatches (discussed in detail below). Typically, an amplification product useful in the present method should be at least about 40-45 nucleotides in length, with at least about 45 nucleotides in length being preferred. While the maximum size of a suitable amplification product can vary widely, and can be up to 1000 nucleotides in length or larger (and therefore explicitly includes any size amplification product between 40 or 45 and 1000 nucleotides or more, in whole integer increments), in one aspect of the invention, the amplification product is between about 40 or 45 and about 500 nucleotides in length, or in another aspect, is less than about 300 nucleotides in length, or in another aspect, is less than about 100 nucleotides in length, or in another aspect, is less than about 90 nucleotides in length, or in another aspect, is less than about 80 nucleotides in length, or in another aspect, is between about 70 to about 80 nucleotides in length. The Examples illustrate the production and successful use of amplification products that are 208 nucleotides in length and amplification products that are 82 nucleotides in length. Generally, and without being bound by theory, the inventors believe that smaller amplification products (e.g., in the range of 45-100 nucleotides) will perform better in the method of the invention by allowing better resolution of melting temperatures at later stages in the method and more robust amplification of low-quality genomic DNA samples. This phenomenon may be a result of the fact that shorter amplicons will have less potential for higher order structures that could compete with or block probe binding.

In one aspect of the invention, once the amplification product is produced, it may optionally be purified (or partially purified) from other components used in the amplification reaction. Typically, this step, if used, is performed as part of, or prior to, the step of isolating single-stranded polynucleotides from double-stranded polynucleotides (described below). This step is optional, and may be modified or eliminated depending on the amplification procedure used, but in general, the use of this step, may enhance the removal of components that would otherwise inhibit or interfere with subsequent steps, and is expected to also enrich for the target polynucleotide-containing amplification product, which is useful for the later detection steps. The amplification product can be purified or partially purified using any suitable method. A preferred method is one that is rapid and/or can be adapted to a high-throughput or automated protocol. Suitable purification methods include, but are not limited to, silica membrane spin columns, size exclusion spin columns, other purification columns, gel extraction purification, enzymatic digestion of short nucleotides, exonuclease digestion, and the like. The Examples demonstrate the use of spin columns and gel extraction purification, and illustrate the tolerance of the method of the invention to a variety of techniques. Isolation of Single-Stranded Polynucleotides

Once an amplification product is produced and purified (or partially purified), the method of the invention includes a step of isolating single-stranded polynucleotides from the amplification product. Using most amplification procedures, the resulting product will be a double-stranded polynucleotide. The final steps in the method of the invention (described below) involve the hybridization of a probe to one strand of the amplification product, and the measurement of the melting temperature of hybrids formed thereby. However, the present inventors have discovered that, in order to properly and accurately detect the target sequences as described herein, it is important to isolate the single- stranded template to which the hybridization probes will bind from the other strand of the amplification product. This is illustrated in the Examples section, where a comparison of the use of double-stranded amplification product to single-stranded amplification product in the method shows that generation of a single-stranded polynucleotide is necessary for accurate genotyping. More specifically, the example demonstrates that the association between the template and its complementary strand within the double-stranded polynucleotide (amplification product) will generate a much higher signal than the association between the template and the hybridization probe. In addition, by using double-stranded polynucleotides for the final steps of the method, less pronounced melting curves are generated for the template -probe hybrid as compared to hybrids formed using a single-stranded polynucleotide template. Therefore, a step that isolates a single-stranded polynucleotide template from the amplification product is necessary for accurate genotyping using the method of the invention. If the amplification process is such that only a single-stranded template is generated (either a sense or an anti-sense strand), then the isolation step could be eliminated. A single-stranded polynucleotide can be isolated from the amplification product using any suitable method for separating and isolating nucleic acids from other nucleic acids or materials, and will typically include a process that allows for the isolation of the single-stranded polynucleotide in conjunction with a denaturing process to separate the target single-strand from its complementary strand. Thus, it is useful to bind the desired template to a substrate or other capturing moiety that can be used to isolate the single- stranded template. In one aspect, this step of the method is conveniently accomplished if, for example, a primer with a selectable moiety was used to produce the amplification product during the amplification step (discussed above). In this case, such a selectable moiety can now be used to "select" or isolate and bind the polynucleotide with the moiety incorporated therein, and then the single-stranded polynucleotide can be separated from its complementary strand that does not contain the selectable moiety, e.g., by denaturing the strands. For example, a polynucleotide incorporating a selectable moiety (e.g., biotin by way of example) can be bound to a binding partner for the selectable moiety (e.g., streptavidin in the case where biotin is the selectable moiety). The binding partner could be coated onto or incorporated into a substrate, including an immobilized substrate or a substrate that can be immobilized (e.g., a magnetic bead). Once the polynucleotide is bound, which will still be in a double-stranded complex, the complex is exposed to denaturing conditions that are sufficient to separate the double-stranded polynucleotides into single strands, but that do not disassociate the selectable moiety from its binding partner. In this way, the non-bound strand can be washed away, leaving the single- stranded polynucleotide (the desired template for the hybridization step) bound to a substrate via the complex of the selectable moiety and the binding partner. An amplification product can be bound to the substrate or other capturing moiety by any suitable method and/or any combination of reagents that allows the strands to be separated and the single-stranded polynucleotide template to be isolated. The only qualification of such method and reagents is that they do not substantially interfere with the hybridization of a probe to the single-stranded polynucleotide in the next steps of the method which include detection of a detectable label used during the melting curve analysis. In addition, the concentrations of these reagents can easily be optimized to maximize the downstream steps of the method, including the signal obtained in the melting curve analysis. An example of optimizing magnetic bead concentration, for instance, is illustrated in the Examples section. Other strategies may include the use of agarose beads, Sepharose® beads, latex beads, etc. In addition, one can couple the polynucleotide to a different substrate, such as a plate or tube directly using various binding ligand/partners.

As discussed above, in order to isolate a single-stranded polynucleotide, immobilization of one or the other strands of the amplification product can be bound to a substrate, typically using an intermediary binding partner (e.g., the complex of a selectable moiety in the polynucleotide and a binding partner on the substrate). Such a substrate can include any suitable substrate for immobilization of a nucleic acid molecule (polynucleotide) or a reagent used to capture or isolate such nucleic acid molecule. Such a substrate can include, but is not limited to, any solid support, such as any solid organic, biopolymer or inorganic support that can form a bond with the nucleic acid molecule or a reagent that binds to the nucleic acid molecule without significantly effecting the downstream steps in the method of the invention. Exemplary organic solid supports include polymers such as polystyrene, nylon, phenol-formaldehyde resins, and acrylic copolymers (e.g., polyacrylamide). Exemplary biopolymer supports include cellulose, polydextrans (e.g., Sephadex®), agarose, collagen and chitin. Exemplary inorganic supports include glass beads (porous and nonporous), magnetic beads, stainless steel, metal oxides (e.g., porous ceramics such as ZrO₂, TiO₂, AI2O3, and NiO) and sand. In one embodiment, beads, such as magnetic beads, are coated with or bound with a binding partner for a selectable moiety that has been incorporated into one strand of the amplification product.

To denature a double-stranded polynucleotide amplification product into single strands, any suitable denaturing protocol may be used. As mentioned above, the denaturing conditions should be sufficient to denature (separate) the polynucleotide strands, but should not disrupt or interfere with the ability to isolate the target single- stranded polynucleotide (e.g., if the single-stranded polynucleotide is bound to a substrate, the denaturing conditions should not cause the polynucleotide to become dissociated from the substrate. Denaturing conditions for double-stranded polynucleotides are known in the art. One particularly useful denaturing condition for use in the present method is to expose the double-stranded polynucleotide to denaturing buffer comprising between about 0.01 M and about 0.5 M NaOH, and in one embodiment, to about 0.15M NaOH. The denaturing conditions may also include from about IM urea to about 8M urea, from about 10% formamide to about 60% formamide, and/or any other reagents that can break the hydrogen bonds in double-stranded DNA. In addition, denaturing conditions may include the application of increased temperature sufficient to break the hydrogen bonds. In general, the stringency of the reagents in the denaturing buffer can be reduced as the temperature of the denaturing conditions increase.

In one aspect of the invention, if a selection moiety was bound to or incorporated into the single-stranded polynucleotide, the method may, if desired, include a step which removes the selection moiety from the polynucleotide after denaturation. This step is typically not necessary, but could be useful, for example, should the selection moiety interfere with the downstream steps of the assay. Probe Hybridization Step

Following the isolation of a single-stranded polynucleotide from the amplification product, the genotyping method of the invention includes a step of contacting the single stranded polynucleotide with a hybridization probe and with a label that detects hybridization of a single-stranded polynucleotide and the hybridization probe. This step is conducted under conditions sufficient to cause the single-stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide, and the conditions are additionally those that are suitable for performing a melting temperature analysis (described below). In addition, conditions and procedures for probe hybridization to a target sequence that are described in detail below can be applied to the use of the specific oligonucleotide probes described herein in other methods, including without limitation the use of these probes to hybridize to target sequences in another genotyping method.

As used herein, a "hybridized polynucleotide" (also referred to as a "hybrid") refers to a polynucleotide that is formed by the hybridization (base pairing, binding) between two single-stranded polynucleotides, which in this case, are the hybridization probe (defined below) and the single-stranded polynucleotide isolated in the prior step of the method. For purposes of this invention, a hybridization probe that is not an oligonucleotide per se, but which is a nucleic acid binding molecule, e.g., PNA, RNA, or LNA, is also included in the definition of a hybridized polynucleotide when it binds to a single-stranded polynucleotide. The degree of hybridization between the two members of the hybrid need not be perfect to be deemed a hybridized polynucleotide. More particularly, in one aspect of the invention, a hybridized polynucleotide can include a "perfect hybrid" (also referred to herein as a "matched hybrid" or derivatives of these terms), where the members of the hybrid are fully complementary to each other over the full length of the shorter of the two members {i.e., the probe, in the case of the probe- polynucleotide hybrid). Those of skill in the art understand that fully complementary nucleotides form base pairings that are non-covalently connected via two or three hydrogen bonds. However, reference to a hybridized polynucleotide can also include "imperfect hybrids" (also referred to as "partial hybrids", "mismatched hybrids", or derivatives of these terms), wherein the members of the hybrid differ in sequence by at least one nucleotide (such that at least one of the nucleotides of one member is not complementary to the corresponding nucleotide of the other member). A single inconsistency between the two strands of a hybrid will make binding between them more energetically unfavorable, and thus, the melting temperature (T_m) of an "imperfect" or "mismatched" hybrid will be lower than that of a "perfect" or "matched" hybrid.

The amount of single-stranded polynucleotide required for this step of the invention can be quite small (at least about 20 ng of polynucleotide), and generally ranges from about 20 ng to about 500 ng of polynucleotide per reaction condition (i.e., per hybridization probe), and preferably from about 20 ng to about 300 ng, and may expressly include any amount in between either range, in 1 ng increments. In one aspect, it is preferable to keep the concentration of the single-stranded polynucleotide template equal to that of the probe or preferably lower than that of the probe. An advantage of the method of the invention is that the amplification step will generate large amounts of polynucleotide template from a very small starting nucleic acid sample, providing sufficient template to run a large number of hybridization reactions, thereby allowing for the simultaneous screening of multiple genes, multiple selected sites within a single gene, and/or multiple mutations or variations within a selected site, all in a single assay and all from a single, original patient nucleic acid sample. Moreover, because the polynucleotide that is used in the hybridization step comes from a single amplification reaction with the same set or sets of primers, regardless of how many different probes are tested, variability among hybridization wells is decreased because the template is homogeneous, which provides enhanced consistency and reproducibility to the method. The hybridization probe is configured (e.g., has a particular sequence, by design or selection) to hybridize to a nucleic acid sequence spanning the selected site of the target polynucleotide. The hybridization probe may have a nucleic acid sequence at the selected site corresponding to (i) a target sequence, or alternatively, to a (ii) a non-target sequence. The inclusion in the assay of probes that binds to one or more non-target sequences (e.g., a wild-type sequence or a sequence for a different variant or mutation) provides not only a control for the experiment, but as discussed above, can also reveal information about the presence of sequences that were not fully inhibited during the amplification step, as well as information about the presence of unexpected or extremely rare sequence variations within the selected site that are not being specifically screened for in current test. This is illustrated in the Examples section and will be discussed below in more detail.

According to the present invention, a "hybridization probe" (also referred to as simply, a "probe") is a nucleic acid binding agent that is typically used to identify a target nucleic acid sequence in a sample by hybridizing to such target nucleic acid sequence under stringent hybridization conditions. A suitable nucleic acid binding agent can include, but is not limited to, oligonucleotides, PNA, RNA, LNA, and any other nucleic acid binding agent. For use in the present method, however, it is desirable to use a probe that can form a hybrid that can be detected, when hybridized to a nucleic acid template, by a double-stranded nucleic acid label, such as a double-stranded DNA binding dye. A probe typically binds to its target because the probe has a structure or sequence that is complementary to at least a portion of the target sequence.

When the probe is an oligonucleotide, the probe typically ranges in size from about 8 nucleotides to several hundred nucleotides in length (the equivalent lengths can also be ascribed to PNA probes, although these are most typically smaller than 30 nucleotides in length). For use in the present invention, smaller probes are desirable, which improves the ability to discriminate matched probes (those that will form perfect or matched hybrids with the template) from mismatched probes (those that will form imperfect, or partial/mismatched hybrids with the template) using the method of the invention (discussed below). Preferably, the hybridization probe is between about 10 and about 50 nucleotides in length (or the equivalent, if not an oligonucleotide), and in one aspect, between about 10 and about 45 nucleotides in length, and in one aspect, between about 10 and about 40 nucleotides in length, and in one aspect, between about 10 and about 35 nucleotides in length, and in one aspect, between about 10 and about 30 nucleotides in length, and in one aspect, between about 10 and about 20 nucleotides in length (or less than about 20 nucleotides in length). In one aspect, the hybridization probe is about 15 nucleotides in length. In one aspect, the hybridization probe is about 16, 17, 18, 19, or 20 nucleotides in length. For melting curve analysis, which is the detection step of the method, the probes should be designed so that the expected T_m of probe-template hybrids, whether matched or unmatched, is within a useful range for detection using this type of analysis, which is most typically between about 60⁰C and about 70⁰C, although the range could be expanded to lower and higher T_ms (e.g., 50⁰C to 85°C, including any whole degree increment in between), if desired. Examples of various specific probes that can be used to detect mutant and wild-type ras at different selected sites are provided herein, but the invention is not limited to the use of these probes.

It is not necessary that all probes in a given genotyping method be of the same size, even when the probes are members of a plurality of probes targeting the same selected site of a target polynucleotide. Indeed, it is an element of the invention to optimize the size of the probe to hybridize to its target sequence. By way of illustration of this point, a plurality of probes may include five probes, each of which binds to a different target sequence at a selected site of a target polynucleotide (e.g., a different mutation in codon 12 of ras). Three of the five probes may be 15 nucleotides in length, while the two are 18 nucleotides in length, the sizes selected because for each probe, it optimizes the ability to distinguish (using Tm) between a match and a mismatch when using that particular probe.

As mentioned above, a hybridization probe used in the method of the invention has a nucleic acid sequence, or other chemical composition, that binds to a complementary sequence (fully or partially) in the single-stranded polynucleotide template that spans the selected site. Typically, in the method of the invention, a plurality of probes is provided, each of which binds to a different sequence spanning the selected site. Some probes may bind to a target sequence {e.g., a particular mutant sequence in a target polynucleotide that the method user would like to detect), and some probes may bind to a non-target sequence (e.g., a wild-type sequence at the selected site). Accordingly, the probes can be used to identify the presence or absence of one or more target sequences in the nucleic acid sample from a given source (e.g., a patient tumor sample). Several specific probes useful in the methods of the invention are described elsewhere herein, including in the Examples.

Probes used in the method of the invention need not be labeled, as the invention includes the use of a separate detectable label, which is described below. This is an advantage of the invention method, because the method allows for the use of multiple different probes with only a single label, thereby enabling detection of multiple target sequences without placing a limitation on the multiplexing ability of the assay due to a need for separate labels for each condition.

As discussed above, the hybridization probe or probes are contacted with the single-stranded polynucleotide under conditions sufficient to cause the single-stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide. As used herein, hybridization conditions refer to standard hybridization conditions (temperature, salt concentration, etc.) under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly referred to herein as "Sambrook"). Sambrook, ibid., is incorporated by reference herein in its entirety. In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al, 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety. One of skill in the art can use the formulae in Meinkoth et al., ibid, to calculate the appropriate hybridization and wash conditions to achieve particular levels of nucleotide match and/or mismatch. Such conditions will vary, depending on whether DNA:RNA hybrids, DNA:DNA hybrids, PNA:DNA hybrids or RPN:RNA hybrids, etc., are being formed. Alternatively, T_m can be calculated empirically as set forth in Sambrook, supra, pages 9.31 to 9.62, with respect to the 1989 addition. As discussed above, preferably, the probes and hybridization conditions are selected so that the expected T_m of probe-template hybrids, whether matched or unmatched, is within a useful range for detection using this type of analysis, which is generally between about 50⁰C and about 85°C.

According to the present invention, sufficient conditions to cause the single- stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide can be determined using the guidance provided above and based on the size of the template and probe. In one aspect, sufficient conditions include the use of a hybridization buffer (also referred to herein as a melting buffer or melting curve buffer) that has a salt concentration of between about 0.1X SSC and about 2X SSC, and expressly including any concentration in between, in 0.1X increments (e.g., 0.1X, 0.2X, 0.3X, 0.4X, 0.5X...). The present inventors have discovered that in some probe-template combinations, by using lower salt conditions during the hybridization and melting curve analysis, better discrimination between matched (perfect) and unmatched (imperfect) hybrids can be achieved. The salt concentrations can be optimized for each probe-template combination.

This step of the method also involves the use of a label that detects hybridization of a single-stranded polynucleotide and the hybridization probe. Labels may also be used with the specific probes described herein when such probes are used in other target sequence detection methods, such as other genotyping methods. Preferably, the label is not incorporated into or attached to the hybridization probe, which is an advantage of the present invention, since multiple probes can be used in the method with only a single detectable label, avoiding scenarios where there are limitations on multiplexing analyses due to availability of a finite number of dyes or other labels. A suitable label is any label that will detect hybridization between two polynucleotides (i.e., when the polynucleotides are in the double-helical state, also referred to as detecting double-stranded nucleic acids), but does not detect single-stranded polynucleotides (i.e., the label differentially binds to double-stranded and single-stranded polynucleotides, or otherwise produces a different signal based on the quantity of double-stranded nucleic acids present). In one aspect, the label is a double-stranded nucleotide binding agent, which is defined herein as any agent (compound, molecule, etc.) that binds to double-stranded nucleic acids. In one aspect, such a binding agent can be a double-stranded nucleotide intercalating agent, which is defined herein as any agent (molecule, compound, etc.) that intercalates into double- stranded nucleic acids. Such a label can be detectable by any suitable means, but in one aspect, is detectable by emission of fluorescence. In one aspect, such binding agents include double-stranded nucleic acid fluorescent dyes. A variety of double-stranded nucleotide binding dyes are known in the art. Examples include, but are not limited to: SYBR^® Green I (an asymmetrical cyanine dye that binds to double-stranded nucleotides; Invitrogen); SYBR^® GreenER, SYTO^® 9; SYTOX^®, etc. Ethidium bromide is an example of an intercalating agent. In addition, a variety of cationic cyanine dyes exhibit high affinity, sequence-independent binding to PNA-containing hybrids, and can be used when PNA is the hybridization probe (see, e.g., Smith et al, 1999, J. Am. Chem. Soc. 121 :2686- 2695, and Dilek, et al., 2005, J. Am. Chem. Soc, 127:3339-3345). Useful dyes also include any dyes that can be tracked using UV or visible light. Melting curves can be generated for these dyes by looking at the absorbance over time and temperature. Such dyes show a shift in the wavelength they absorb when bound to double-stranded species, and a melting curve can be generated at this wavelength. Various cationic cyanine dyes, such as those described above, are an example of such dyes. U.S. Patent No. 7,387,887 also describes dyes that are useful in a method of the invention. The use of two different dyes is illustrated in the Examples. In general, the concentration of dye used in this step can be optimized by simply running a range of concentrations of the particular dye to be used in a melting curve analysis using a matched probe and single-stranded polynucleotide template, for example, and selecting the optimal concentration for visualization of melting curves. Typically, the concentration of dye will be between 1 μM and 10 μM, including any concentration in between, although the concentration can be adjusted by the user of the method according to the dye, probes, templates, and hybridization conditions. An example of optimization of the dye concentration is provided in the Examples.

The method of the invention is highly flexible and is amenable to multiplex genotyping strategies (i.e., screening for multiple different target sequences concurrently). Therefore, the method of the invention is very adaptable to high throughput and automated screening. Indeed, because each probe and single-stranded polynucleotide hybrid will have a different T_m, one can conceivable screen for two or more different sites within the same gene, or even two or more different genes, within the same sample aliquot (well or tube, etc.). A variety of different assay designs can therefore be envisioned for the method of the invention, each of which incorporates multiplexing, although the invention is not limited to these designs.

In one embodiment of the invention, the hybridization step includes distributing the single-stranded polynucleotides generated in the prior step into two or more aliquots (and up to as many aliquots as possible given the amount of polynucleotide obtained from the earlier steps). Each aliquot is then contacted with (mixed, combined with, etc.) the detectable label that binds to double-stranded hybrids and with one of a plurality of hybridization probes to form a hybridized polynucleotide. In this example, each hybridization probe in the plurality of probes has a different sequence at the selected site as compared to the other probes in the plurality, and each aliquot is contacted with a single different hybridization probe. While the single-stranded polynucleotides can be divided into as many aliquots as desired (or up to the limit of the amount of polynucleotides available), each sample reaction, whether it be in a tube, a well, or other chamber, this assay design will test the ability of one probe to hybridize to the polynucleotide per aliquot. Each probe can detect a different target sequence or non-target sequence at a single selected site of a target polynucleotide, or alternatively, the assay can be designed so that the plurality of probes detect target or non-target sequences in two or different selected sites of the same target polynucleotide or gene, or even different target sequences in different genes (or combinations of all of the foregoing). In the case of screening for different selected sites of the same target polynucleotide or different target sequences in different genes, of course, these designs are dependent on the amplification step including the appropriate additional primer pairs to amplify target polynucleotides (creating appropriate amplification products) for each of the desired selected sites and/or different genes or portions thereof (discussed previously herein). The method of the invention is flexible such that a variety of target sequences can be detected in a single assay. The detection step following the hybridization (by melting curve analysis) is straightforward in this scenario, since the user knows which primer is in which reaction aliquot, and can compare T_ms from those target or non-target sequences that bind to the same selected site in order to detect the genotype of the sample at that site. This step is described in more detail below and is illustrated in the Examples.

In another embodiment, the assay can be designed so that the hybridization step again includes distributing the single-stranded polynucleotides generated in the prior step into two or more aliquots, up to as many aliquots as possible given the amount of polynucleotide template that is available. In this design, however, each aliquot is contacted with the detectable label with two or more of a plurality of hybridization probes to form a hybridized polynucleotide. In this example, each hybridization probe in the plurality of probes again has a different sequence at the selected site as compared to the other probes in the plurality, and each aliquot is contacted with two or more different hybridization probes. However, within a single aliquot, the two or more probes should bind to different selected sites of the same or different genes, and in this case, the Tm of a hybrids formed between one matched probe and its template must be sufficiently different than the Tm formed between the other matched probe(s) and its template(s), so that discrimination between the matched hybrids is clear and so that one probe can be distinguished from the other. Generally, under these conditions, the Tms of two different perfect hybrids should be separated by at least 10⁰C or more. Alternatively, if one probe is an oligonucleotide probe and the other probe is a PNA probe, for example, one may be able to discriminate among hybrids by selection of the appropriate dye for each probe type. For example, PNA-containing hybrids do not bind well to all DNA-binding dyes. However, there are dyes that are known to bind well to PNA-containing hybrids (e.g., cationic cyanine dyes), and accordingly, one could mix an oligonucleotide probe and a PNA probe in the same hybridization aliquot and by using dyes specific for each hybrid, identify the Tms corresponding to each probe.

By using these variations on the design of the assay, the capacity of the multiplexing ability of the assay is therefore expanded, because each reaction in the hybridization step provides information about two or more target sequences. It is envisioned that within the same well, one could include probes that detect different target or non-target sequences in two or different selected sites of the same gene or target polynucleotide, or different target sequences in different genes. Again, the appropriate amplification products will be generated in the first step of the method to provide the appropriate template in the mixture of single-stranded polynucleotides. A probe with a given specificity may even be used more than once in different aliquots, but perhaps in combination with different sets of other probes in each aliquot. Detection of the genotype in this assay design is more complex than in the prior design, but nonetheless may be achieved, because primer-template hybrids spanning different selected sites or in different genes will have different melting temperatures. By the appropriate use of controls, the differentiation of matched and unmatched hybrids at different selected sites can be determined.

In this manner, one can screen for any of a number of mutations or variations in a gene at any number of sites, and one can also screen for mutations or variations in several different genes, all starting from the same patient sample and the same amplification reaction, and all within just a few hours. An extraordinary amount of information about a patient genotype can be gained from a single assay, including information regarding what type of disease or condition a patient has, what biological pathways are impacted by variations in the patient genome, whether a prognosis can be determined based on the genotype (e.g., a cancer that is more or less likely to metastasize), and importantly, what specific therapeutic protocol will be most beneficial to the individual. Indeed, the method of the invention provides a genotyping result that allows a clinician to personalize the therapy for the patient based on that genotype, and that can be used in conjunction with selection of treatment for a particular patient. Melting Temperature Determination Step

Once the probes and single-stranded polynucleotides are combined with the detectable label and other appropriate buffers, a melting curve of each aliquot is generated, and the melting temperature, or T_m, is determined. Melting temperature has been defined previously herein. Melting curves are typically generated by measuring the detectable label (fluorescence, in the case of most double-stranded DNA binding dyes) as temperature is increased step-wise over a change of about 20-40⁰C, typically starting at about 40-50⁰C and proceeding up to about 90⁰C. Using a fluorescent dsDNA binding dye as an example, at lower temperatures, the hybridized polynucleotide produced by hybridization of the probe and single-stranded template will have incorporated the binding dye, and since it is a double-stranded DNA binding dye, the dye will fluoresce. As the temperature increases, the strands of the hybrid will begin to dissociate (melt) into single strands and as this happens, the amount of fluorescence emitted from the dye will decrease, since the dye only fluoresces when bound to a double-stranded nucleic acid. In the case of a fluorescent dye, the raw fluorescence is plotted against the temperature as it is collected by the instrument. The melting temperature is often defined as the temperature where half of the nucleic acid species being studied exists in a double-stranded (hybridized) conformation, while the remaining half has undergone dissociation to form two single strands of DNA. The melting point can be accurately approximated experimentally by looking at the inflection point of the derivative (dF/dT) of the raw data. These derivative curves are herein referred to as "melting curves". In the case of the examples herein, the data was collected and analyzed on a real-time PCR device, capable of both thermal cycling and capture of the fluorescence data. Any thermal cycler with the ability to measure fluorescence could alternatively be used. Additionally, melting profiles can be generated on an appropriate device using UV and visible absorbance should an appropriate label be included in the method. Software is readily available that converts the raw data to derivative plots and identifies the point(s) of inflection for the melting curves, which represent the reported T_m values. In this step of the method, the T_m is detected for each of the hybridized polynucleotides being tested. Melting curves and derivatives are generated, and the data can then be analyzed to detect the target sequences, or absence thereof, at the selected site or sites. Detection of Target Sequences Step Once the T_m has been generated for all of the hybrids in the assay, the T_ms for each of the hybridized polynucleotides formed with probes spanning the same selected site are compared to one another, in order to identify whether any of the hybrids is a perfect hybrid based on T_m, thereby identifying the presence of a target sequence in the nucleic acid sample. Perfectly hybridized polynucleotides (matched hybridized polynucleotides, or those that are fully complementary over the full length of the shorter sequence), will have a significantly and detectably higher T_m than hybridized polynucleotides formed between imperfectly hybridized polynucleotides (mismatched hybrids, or hybrids formed with a probe having a nucleic acid sequence that is different from the single-stranded polynucleotide at the selected site by at least one nucleotide). Typically, the difference in T_m between matched and mismatched hybrids at the same selected site will be between about 2° and 25°C, and in one aspect, between about 2°C and about 10⁰C, and is preferably at least 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10⁰C different. In multiplexing strategies, differences in melting temperatures may be increased above 10⁰C. In some circumstances, a probe may generate two or more T_m values (visualized as "peaks" when the derivative is calculated and graphed), where one of the T_m values is usually higher than that generated by the other probes spanning the selected site, and where the other of the T_m values is very similar to that generated by the other probes. Such a result can indicate that the given probe producing two T_m values is hybridizing to a matched template and also to a mismatched template that is present in relatively high abundance in the amplification product. This will occur, for example, if the amplification step did not fully inhibit the amplification of the non-target sequence, or if there are two or more different target sequences at the selected site that occur within that patient nucleic acid sample. The identity of the probes will distinguish between these different possibilities.

To more clearly illustrate this point, although the invention is not limited to this example, in the case of genotyping a tumor sample, one may wish to detect the presence or absence of a mutation in the ras gene. In this particular application of the invention, it is noted that the amount of wild-type ras DNA will typically greatly outnumber the amount of mutant ras DNA in a tumor sample, due to the presence of stromal cells in the patient tumor sample, and/or the presence of a mutation in only one of the two chromosomal copies of the gene present in each cell (heterozygous), for instance. Therefore, mutated ras is usually considered to be a rare or underrepresented sequence in a genomic DNA sample, and is ideal for detection using the method of the invention. This is because the amplification step can be designed to amplify a portion of the ras gene (a selected site), while specifically inhibiting amplification of the wild-type ras DNA (e.g., by using a PNA that clamps the wild-type sequence and inhibits its amplification), thus enhancing amplification of any mutated ras DNA in the patient sample. Now suppose that the patient sample does contain mutated ras at codon 12 (a selected site), for example, and further suppose that inhibition of the wild-type amplification was incomplete. Accordingly, when determining the melting temperature of the hybrids formed between various probes targeting codon 12 mutations or the wild-type sequence at codon 12, those reaction wells that contained a probe that is fully complementary to the wild-type sequence (the non-target sequence), and those reaction wells that contained a probe that is fully complementary to the patient's particular mutation at codon 12 (a target sequence), should each produce two Tm values, one that is higher as compared to all of the other probes due to perfect hybrids formed with the template (which contains a wild-type species and one mutated species) and one that is similar to the Tm of all of the other probes due to mismatched hybrids formed with the template. This does not render the method unusable for this patient; rather, one can determine the source of the second peak through the use of multiple probes, and the T_m created by the matched hybrids will in any event be significantly higher than the mismatched hybrids. In the case where only one probe forms two different T_ms, then it can be deduced that the sample contains a species of the genotype detected by the probe at the higher T_m and that the sample contains a second, unknown variation at or near this selected site, which could be identified, if desired, through the use of additional probes or another method such as sequencing. For example, in this manner, the method of the invention has detected the presence of a mutation at codon 11 of the ras gene in a patient sample while screening for mutations at codon 12, where a probe specific for the wild-type sequence identified a second hybrid that was not accounted for by the mutant probes for codon 12. While the identity of the codon 11 mutation was not known from the genotyping method of the invention, its presence was noted and was confirmed later by sequencing the genomic DNA through this region of the gene.

The Examples, while not intending to be limiting in any manner, illustrate the use of various aspects of the method of the invention. For instance, Example 1 describes one protocol within the scope of the invention in detail, including the identity of primers, PNA molecules and probes useful in such method. Uses of the Method of the Invention

Having described the steps of the genotyping method in detail, as well as the individual steps of amplification of target sequences or detection of target sequences using the specific primers, PNA and probes provided herein, it can now be appreciated that the methods of the invention have a wide variety of applications, and can perform complex multiplex genotyping in a straightforward, rapid and consistent manner.

The target sequences that can be detected using the genotyping method of the invention include any target sequence that contains a variation as compared to another sequence at the same site. The method is useful for detecting virtually any mutation or variation in a sequence, including substitutions, deletions, insertions, and derivatizations of nucleotides in a given sequence. There is no limitation on the number of individual substitutions, deletions, insertions, and derivatizations in a given selected site that can be detected, other than those placed on the ability to amplify a target polynucleotide containing that selected site, and to differentiate melting temperatures of matched and mismatched hybrids formed using probes that bind to that selected site. In general, the method will be used to detect less than about 10 nucleotide variations in a given selected site, and more typically, about 1, 2, 3, 4 or 5 variations at that site. Point mutations and mutations including 2 or 3 nucleotide variations are readily detectable using the method of the invention.

Mutations in virtually any target polynucleotide and accordingly, virtually any gene, can be detected using the method of the invention. However, as discussed previously herein, the method is especially valuable for detecting mutations or variations that are rare or underrepresented in a nucleic acid sample, as many genotyping methods cannot detect such mutations or variations. In one embodiment, the method of the invention is used for detecting mutations in a gene associated with a disease or condition in a patient. In another embodiment, the method of the invention is used for detecting mutations in a gene associated with a particular biological pathway in an individual. In another embodiment, the method of the invention is used to screen for chromosomal rearrangements that have generated new junctional DNA sequences (e.g., the Bcr-Abl fusion in chronic myelogenous leukemia). In another embodiment, the method of the invention is used to screen for escape mutations that occur as a result of exposure of a subject to small molecule therapy or immunotherapy. In addition, the method of the invention can be extended to infectious diseases. For example, the efficiency of replication for certain viral diseases such as Hepatitis B virus (HBV), Hepatitis C Virus (HCV) and Human Immunodeficiency Virus- type 1 (HIV-I) depends upon a variety of factors including: 1) host genetic makeup and frequency of certain mutations (Jopling and Norman, 2006, supra; Yu and Lichterfeld, 2007, supra), and; 2) the presence of 'escape mutations' in the genomes of the viruses themselves which render the viruses refractory to drug efficacy or recognition by the immune system (Wolfl, 2008, supra; Shibata, 2007, supra; Sloan, 2008, supra). In this embodiment, a patient sample, such as a blood or tissue sample, is tested in the method of the invention to detect the presence or absence of viral RNA or DNA, and particularly, to test for viral RNA or DNA that has mutated.

In yet another embodiment, the method of the invention is used to genotype fetal DNA. such as to determine red blood cell antigen status, as well as other potential conflicts between fetal and maternal genomes that may result in disease, or a variety of genetic mutations that may be identified in the fetus. Fetal DNA is difficult to obtain and quantities are limited, and so the method of the invention is expected to be readily adaptable to multiplex genotyping of this source of DNA.

Examples of genes that are associated with diseases or conditions and/or that may have mutations or variations which it would be desirable to detect using the method of the invention are known in the art and include genes encoding a variety of growth factors, growth factor receptors, signal transducers, transcription factors, tumor suppressors, and programmed cell death regulators. Such genes, include, but are not limited to, the genes encoding: Ras, EGFR, BRAF, MEK, BCR-AbI, plO, p53, JAK2 kinase, HER2/neu, RBI, INK4a, APC, MLHl, MSH2, MSH6, WTI, BRCAl, BRCA2, VHL, N-myc, C-myc, EWS, BCL-2, NFl, and NF2.

In one aspect of the invention, a target polynucleotide to be detected using the genotyping method is at least a portion of a ras gene (H-Ras, N-Ras or K-Ras). The nucleotide and amino acid sequence for a variety of Ras family members are well known in the art, and so, using the teachings provided herein, primers and various other nucleic acid binding agents used in the methods of the invention can be produced. SEQ ID NO:1 is the nucleic acid sequence encoding human K-ras. SEQ ID NO:1 encodes human K-ras, represented herein as SEQ ID NO:2. SEQ ID NO:3 is the nucleic acid sequence encoding human H-ras. SEQ ID NO:3 encodes human H-ras, represented herein as SEQ ID NO:4. SEQ ID NO: 5 is the nucleic acid sequence encoding human N-ras. SEQ ID NO: 5 encodes human N-ras, represented herein as SEQ ID NO:6. SEQ ID NOs: 1-6 are representative of "wild-type" Ras sequences. As used herein, unless otherwise stated, reference to "Ras" or "ras" can refer to any Ras or ras, respectively, including K-Ras, H-Ras, or N-Ras (or k- ras, h-ras, or n-ras). As discussed previously herein, Ras is an example of an oncogene in which several mutations are known to occur at particular positions and be associated with the development of one or more types of cancer. For example, mutations are known to occur at positions 12, 13, 59, 61 and 76 (codons 12, 13, 59, 61 or 76, with respect to the nucleic acid sequences), and in some cases, more than one mutation will occur at the same codon in a given patient tumor, or more than one mutation will occur at different codons in a given patient tumor. Indeed, the combination of mutations at codon 12 and 76 has been shown to synergize and significantly increase the oncogenicity of a tumor bearing such combination of mutations (see U.S. Patent Publication No. 1007/0224208). At codon 12, mutations that may be detected using the method of the invention include, but are not limited to: G12R, G12V, G12D, G12C, G12S, and G12A. At codon 13, mutations that may be detected using the method of the invention include, but are not limited to: G13D. At codon 59, mutations that may be detected using the method of the invention include, but are not limited to: A59T. At codon 61, mutations that may be detected using the method of the invention include, but are not limited to: Q61R, Q61L, Q61H, and Q61P. At codon 76, mutations that may be detected using the method of the invention include, but are not limited to: E76G, E76K, and E76Q. Using ras as a way to illustrate the flexibility of the method of the invention, and the power to use this method in multiplexing strategies, one can screen, in a single assay, for all of the mutations at codon 12, all of the mutations at codon 13, all of the mutations at codon 59, all of the mutations at codon 61 and/or all of the mutations at codon 76. In addition, if desired, one can also screen for mutations or variations in any one or more selected sites of any one or more additional genes. For example, it may be valuable to detect whether mutations are occurring in any of the genes that are downstream from or otherwise in the same or related biological pathway as the ras gene, such as genes encoding: MEK, ERK, BRAF, RAF, MAPK, P13K, PTEN, and AKT. In one aspect, the method includes detecting mutations in the gene encoding BRAF. A mutation that may be detected using the method of the invention includes, but is not limited to, V600E (see, e.g., Nicolantonio et al., 2008 Nov. 10, J. CHn. Oncol, epub.). This mutation can be detected alone or in combination with any one or more ras mutations or mutations in one or more other genes.

As another example, if screening for the best therapeutic approach to treat a particular cancer that is associated with overexpression of epidermal growth factor receptor (EGFR), it would be highly valuable to detect whether a patient tumor has EGFR mutations and whether the patient tumor has any mutations in the ras gene, as the presence of one or the other may completely redirect the patient treatment. Other examples of multiplex screening will be clear based on the discussion provided herein.

Once the presence or absence of one or more target sequences is detected using the method of the invention, and particularly when such target sequences are associated with a disease or condition, the method can, in one embodiment, include the preparation of a report for a clinician or other party that identifies the target sequences that were identified (or the absence of detection of any of the targets). This information can then be used by a clinician or other party to determine a diagnosis for a patient, to determine a prognosis for a patient, to determine the appropriate therapy to administer to a patient, and to predict the patient's success or outcome with a therapeutic protocol. The clinician may also order the screening for additional target sequences based on the results of the initial screening, which can be rapidly performed, particularly if portions of the amplification product or single-stranded polynucleotide remain after performance of the initial screening. Alternatively, if the clinician or the laboratory of the clinician is directly performing the method of the invention, then the clinician will be able to directly evaluate the results of the method and either perform additional assays to identify other target sequences or combinations thereof, or proceed to a diagnosis, prognosis, or prescription of therapy as described above.

Another embodiment of the invention therefore includes a method of prescribing treatment for a cancer that includes identification of a particular mutation in the DNA of a patient. The method includes the steps of: (a) identifying a mutation in a target polynucleotide of a patient who has cancer by reviewing a report that identifies the mutation, wherein the mutation was detected using the method as described in any of the embodiments herein; and (b) administering to the patient a therapy that is specific for the mutation identified in the report.

Another embodiment of the invention relates to the manufacture of a therapeutic agent or of an assay kit for performing the method of the invention. Since the method of the invention may, in one embodiment, be required or recommended as a prerequisite to administration of a targeted therapy to a patient, the inclusion of package labeling in the manufacture of either the agent or the kit requiring or recommending the use of the other (in combination) is encompassed by the invention. For example, one embodiment includes a packaged medicament that includes (a) a therapeutic agent that is specific for one or more mutations associated with a disease or condition; and (b) package labeling that requires the use of the genotyping method as described herein confirm the presence of the specific mutation or mutations in a patient in conjunction with administration of the agent to the patient. The reverse labeling would apply to packaging for an assay kit (i.e., the assay kit is manufactured to include package labeling listing the therapeutic agents with which use of the assay kit is required or recommended. Similarly, the invention relates to manufacturing methods themselves that require the production and use of such labeling on therapeutic agents or kits of the invention. Reagents, Kits and Systems of the Invention

The present invention includes oligonucleotide primers that are useful for producing amplification products from target polynucleotides that contain a selected site. These primers may be used in any suitable method that requires or would benefit from amplification of a polynucleotide, including but not limited to the genotyping method described herein, a sequencing method, or any other genotyping method known in the art. The primers of the invention may be provided in the form of a kit or library of primers.

This aspect of the invention includes an oligonucleotide primer or homologue thereof, that hybridizes to, and/or is used for amplification of, sequences from exon 2 of ras, and which include codon 12 of ras. These primers are selected from a primer having a sequence comprising or consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, or SEQ ID NO:58. Homologues of these sequences are expressly encompassed by the invention. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above-identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, SEQ ID NO:7 with any of SEQ ID NO:8, SEQ ID NO: 10 or SEQ ID NO: 11; SEQ ID NO:9 and SEQ ID NO: 10; SEQ ID NO:53 and SEQ ID NO:54, or SEQ ID NO:57 and SEQ ID NO:58. Any other combination of forward and reverse primers selected from the above primers is also encompassed by the invention.

This aspect of the invention also includes an oligonucleotide primer or homologue thereof that hybridizes to, and/or is used for amplification of, sequences from exon 3 of ras and which include codon 61 of ras. These primers are selected from a primer having a sequence comprising or consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:59, or SEQ ID NO:60. Homologues of these sequences are expressly encompassed by the invention. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above-identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, the combination of SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:27 and SEQ ID NO:30, SEQ ID NO:27 and SEQ ID NO:48, SEQ ID NO:27 and SEQ ID NO:56, SEQ ID NO:27 and SEQ ID NO:60, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:29 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:48, SEQ ID NO:29 and SEQ ID NO:56, SEQ ID NO:29 and SEQ ID NO:60, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:47 and SEQ ID NO:56, SEQ ID NO:47 and SEQ ID NO:60, SEQ ID NO:47 and SEQ ID NO:28, SEQ ID NO:47 and SEQ ID NO:30, SEQ ID NO:55 and SEQ ID NO:56, SEQ ID NO:55 and SEQ ID NO:48, SEQ ID NO:55 and SEQ ID NO:60, SEQ ID NO:55 and SEQ ID NO:28, SEQ ID NO:55 and SEQ ID NO:30, SEQ ID NO:59 and SEQ ID NO:60, SEQ ID NO:59 and SEQ ID NO:48, SEQ ID NO:59 and SEQ ID NO:56, SEQ ID NO:59 and SEQ ID NO:28, or SEQ ID NO:59 and SEQ ID NO:30. This aspect of the invention also includes an oligonucleotide primer or homologue thereof that hybridizes to, and/or is used for amplification of, sequences that include codon 600 of braf. These primers are selected from a primer having a sequence comprising or consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:49 or SEQ ID NO:50. Homologues of these sequences are expressly encompassed by the invention. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above-identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, SEQ ID NO:36 and SEQ ID NO:37, or SEQ ID NO:49 and SEQ ID NO:50. Any other combination of forward and reverse primers selected from the above primers is also encompassed by the invention.

This aspect of the invention includes an oligonucleotide primer or homologue thereof that hybridizes to, and/or is used for amplification of, sequences from exon 19 of the gene encoding EGFR. These primers are selected from a primer having a sequence comprising or consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:51 or SEQ ID NO:52. Homologues of these sequences are expressly encompassed by the invention. The invention also relates to primer pairs, which includes, without limitation, the combination of any one of the above-identified primers with any other primer, including any primer not described herein. The invention also relates to primer pairs, which includes any one primer described above with any other one primer described above, including, without limitation, SEQ ID NO:41 and SEQ ID NO:42 or SEQ ID NO:51 and SEQ ID NO:52. Any other combination of forward and reverse primers selected from the above primers is also encompassed by the invention. The present invention also includes PNA molecules that are useful for inhibiting the amplification of target polynucleotides having a non-target sequence at the selected site. These PNA molecules may be used in any suitable method that requires or would benefit from the inhibition of the amplification of a polynucleotide, including but not limited to the genotyping method described herein, a sequencing method, or any other genotyping method known in the art. The PNA of the invention may be provided in the form of a kit or library of PNA molecules.

PNA useful for the inhibition of the amplification of a portion of wild-type ras comprising codon 12 includes a PNA that comprises or consists of the sequence of SEQ ID NO: 17, or a homologue thereof. As discussed above, the PNA of the invention can be used to inhibit the amplification of wild-type ras in conjunction with the use of any amplification primers, including primers designed or developed inside or outside of this invention. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type ras in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds.

PNA useful for the inhibition of the amplification of a portion of wild-type ras comprising exon 3, including codon 61 includes a PNA that comprises or consists of the sequence of: SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, or a homologue thereof. As discussed above, the PNA of the invention can be used to inhibit the amplification of wild-type ras in conjunction with the use of any amplification primers, including primers designed or developed inside or outside of this invention. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type ras in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds. For example, in one aspect, the PNA of SEQ ID NO:26 inhibits amplification of wild-type ras in an amplification reaction in which SEQ ID NO:29 and/or SEQ ID NO:30 are used.

PNA useful for the inhibition of the amplification of a portion of wild-type braf comprising codon 600 includes a PNA that comprises or consists of the sequence of SEQ ID NO:38 or a homologue thereof. As discussed above, the PNA of the invention can be used to inhibit the amplification of wild-type braf in conjunction with the use of any amplification primers, including primers designed or developed inside or outside of this invention. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type braf in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds.

PNA useful for the inhibition of the amplification of a portion of wild-type gene encoding EGFR includes a PNA that comprises or consists of the sequence of SEQ ID NO:43 or a homologue thereof. As discussed above, the PNA of the invention can be used to inhibit the amplification of wild-type EGFR gene in conjunction with the use of any amplification primers, including primers designed or developed inside or outside of this invention. In one aspect of the invention, the PNA is used to inhibit the amplification of wild-type egfr in conjunction with an amplification reaction using primers that overlap with a portion of the sequence to which the PNA binds. The present invention also includes probes that are configured to hybridize to a nucleic acid sequence spanning the selected site of the target polynucleotide. These probes may be used in any suitable method that requires or would benefit from hybridization of the probe to a target polynucleotide, including but not limited to the genotyping method described herein, a sequencing method, or any other genotyping method known in the art. The probes of the invention may be provided in the form of a kit or library of probes.

Probes useful for the hybridization to and detection of a target sequence within exon 2 of ras, which may include wild-type and/or mutated ras sequences, include probes with a sequence comprising: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO : 15 or SEQ ID NO : 16, or a homologue thereof.

Probes useful for the hybridization to and detection of a target sequence within exon 3 of ras, which may include wild-type and/or mutated ras sequences, include probes with a sequence comprising: SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35, or a homologue thereof. Probes useful for the hybridization to and detection of a target sequence within braf, which may include wild-type and/or mutated braf sequences, include probes with a sequence comprising: SEQ ID NO:39 or SEQ ID NO:40, or a homologue thereof.

The present invention also includes a kit (assay kit or genotyping kit) or system for use in practicing of the genotyping method of the present invention. The kit or system includes one or any combination of the following reagents, and in one aspect, includes all of the reagents and/or additional reagents (described below): (a) at least one pair of primers for producing amplification products from target polynucleotides that contain a selected site; (b) one or more reagents that inhibit the amplification of target polynucleotides having a non-target sequence at the selected site; (c) one or more reagents for isolating single-stranded polynucleotides from the amplification product; (d) a label that detects hybridization of a single-stranded polynucleotide and a hybridization probe; and (e) one or more hybridization probes configured to hybridize to a nucleic acid sequence spanning the selected site of the target polynucleotide, wherein each hybridization probe has a nucleic acid sequence at the selected site corresponding either to (i) a target sequence; or (ii) a non-target sequence.

Primers useful for producing amplification products according to the invention have been described in detail above. The kit or system of the invention ideally includes multiple primer pairs, or can be ordered or produced with custom sets of primer pairs, depending on the target sequences to be identified. For example, the primer pairs can include a primer pair for a single selected site of a target polynucleotide or gene, or primer pairs for each of multiple selected sites within a given gene, or primer pairs for one or more selected sites on each of two or more different genes. The primer pairs can be provided in single sets per selected site in order to perform a single amplification round, or if more than one amplification round may or should be performed, then nested primer pairs may be provided for each selected site. Optionally, both a nested primer set and a single round primer set may be provided so that the user can determine whether one or more amplification rounds will be required for a given nucleic acid sample. In one aspect, at least one of the primers in each pair of primers comprises a selection moiety for isolating single-stranded polynucleotides from the amplification product. For example, as discussed above, the selection moiety can include biotin, digoxygenin, any partner of any receptor-ligand pair, an antibody or fragment thereof, FITC, and similar agents.

In the event that an amplification system is used in the method that does not make use of primers as described herein, the this component of the assay kit can include the appropriate reagent or reagents, and particularly any nucleic acid sequence-specific reagents, that are required to perform the amplification step.

Reagents that are useful for inhibiting the amplification of target polynucleotides having a non-target sequence at the selected site have also been described above in detail. Such reagents include, but are not limited to, nucleic acid binding moieties that bind to a non-target sequence at the selected site and thereby inhibit the amplification of a target polynucleotide having the non-target sequence at the selected site. Such nucleic acid binding moieties include, but are not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino oligonucleotides, RNA (and modified RNA), and HyNARNA. Preferably, these reagents in the kit or system are selected to correspond to the primers and hybridization probes that are provided with the kit or system, so that the kit or system provides the complete tools needed to screen for one or more variations at one or more selected sites. Reagents that are useful for isolating single-stranded polynucleotides from the amplification product have been described in detail above. Such reagents include binding agents that bind to any selectable moieties that are included in the primers described above, as well as any substrates or other nucleic acid capturing (binding) agents that either bind directly to nucleic acids or are used as a support for the binding agent. For example, one such reagent, when the primers include biotin, is a streptavidin-coated substrate, such as a streptavidin-coated bead.

Various labels that detect hybridization of a single-stranded polynucleotide and a hybridization probe have been described in detail above. In one aspect, the label is a double stranded nucleotide binding agent. In one aspect, the double stranded nucleotide binding agent is an intercalating agent. In one aspect, the agent is a fluorescent dye. Preferably, a label is chosen that is expected to perform well in conjunction with the hybridization probe(s) that are either included with the kit or recommended for use with the kit. For example, if oligonucleotide probes are provided or recommended for use to detect a particular target sequence in a genomic DNA sample, then a label that is useful for detection of DNA:DNA hybrids should be provided. Alternatively, if PNA probes are provided or recommended for use, then a label that is useful for detection of PNA:DNA hybrids should be provided. In one aspect, more than one type of label can be provided to allow the user to optimize the assay.

Hybridization probes that are useful in the present invention have also been described in detail above. As discussed above, a hybridization probe is a nucleic acid binding agent, which can include, but is not limited to, an oligonucleotide, a peptide nucleic acid molecule, or any derivative or analog thereof, that is used to identify a target nucleic acid sequence by hybridizing to such target nucleic acid sequence under stringent hybridization conditions. The probes will be selected to correspond to the primer pairs that are also provided with or recommended for use with the kit. Preferably, a plurality of probes is provided, which can include probes for a variety of target sequences at the same selected site, a variety of target sequences at different selected sites in the same gene, and/or a variety of target sequences at one or more selected sites on two or more different genes. In one aspect, the user is provided with the probes in free form, so that the exact design of the assay can be determined by the user. For example, the user may wish to expand the multiplexing ability of the assay by including more than one probe in each reaction well or tube, or the user may wish to test one probe condition per reaction well or tube. In addition, the user may wish to use particular combinations of probes to screen for target sequences related to a specific biological pathway of interest, or to screen for target sequences associate with a particular disease or condition. In another embodiment, the hybridization probes are provided in arrays in microplates, with a guide indicating which probe or probes are in which wells of the microplate. In this manner, the user can request or order a particular array of probes and the manufacturer can specify probes sets that are useful for particular applications. This type of kit is also highly amenable to high throughput and/or automation adaptation.

Any of the reagents provided with the kit or system may be present in free form (in separately labeled containers to be aliquoted by the user), or for certain or all reagents, the reagents may be immobilized to or prealiquoted into a substrate such as a plastic dish, beads, a microarray plate, a test tube, a test rod, a test strip and so on.

The kit can also include suitable reagents for performing any of the steps of the method of the invention. For example, the kit can include nucleic acid extraction solutions useful for extracting nucleic acids from a patient sample or other source; suitable reagents for the amplification step (e.g., DNA polymerases, nucleotide triphosphates, buffers for PCR or other amplification reactions); solutions or devices for purification of the amplification product (e.g., spin tubes or gels); denaturing solutions for the single-stranded polynucleotide preparation; buffers for hybridization and melting curve analysis; wash solutions; dilution buffers; and the like. The kit can also include a set of written instructions for using the kit and for interpreting the results. The kit may also include software for analyzing and/or interpreting the results of the melting curve analysis, which may apply to a particular device used for performing the method, or may be applicable to more than one device. The kit can also include positive and/or negative controls, which can include positive and/or negative control templates (e.g., purified single-stranded template for target and/or non-target sequences), and/or control hybridization probes.

In one embodiment, the kit is formulated to be a high-throughput assay. In one embodiment, the kit is formulated to be used in an automated process.

In one embodiment of the invention, the assay kit or system is formulated to be used with a particular device (e.g., a real time PCR device) for performing the amplification step and/or the melting curve analysis. In one aspect, the kit, or a plurality of kits, is provided as part of a complete system with the device. In these embodiments, detailed instructions are provided regarding how to use the kit components with the system, how to validate the device, and how to interpret the results.

The following experimental results are provided for purposes of illustration and are not intended to limit the scope of the invention.

Examples

Example 1

The following example provides an illustration of a genotyping protocol for performing the method of the invention to detect target sequences in codon 12 of the rαs gene. A. Materials and Equipment

• Platinum® Taq DNA polymerase high fidelity (Invitrogen)

• QIAquick® PCR Purification Kit (Qiagen) • Dynabeads® M-270 Streptavidin (Invitrogen)

• DynMag-2™ magnet: (Invitrogen)

• SYBR® Green I, (Invitrogen)

• Oligonucleotides (primers and probes), HPLC purified

Table 1. Primer Sequences

Table 2. Probe Se uences

Equipment: o Real-time PCR machine: (ABI 7500 Fast Real-Time PCR System: Applied

Biosystems Inc.) and Sequence Detection Software vl.4 o PCR machine: (ABI GeneAmp® PCR system 2700, Applied Biosystems

Inc.)

Buffers and solutions o 2x binding and washing buffer (BW): 10 mM Tris-HCL(pH7.5), ImM

EDTA, IM NaCl o Ix Binding and Washing buffer (BW) containing 0.05% Tween®20 o Denaturing buffer: 0.15M NaOH: fresh made before use or -20⁰C stored solution o Melting buffer: 0.5xSSC (lxSSC:0.15M NaCl, 0.015M sodium citrate, pH7.0), 0.5xSYBR® Green I o EB buffer: Tris-Cl, pH8.5

B. Procedure

1) Tumor cell genomic DNA extraction

Incubate tissue or cell sample overnight in TNES lysis buffer and proteinase K solution at 55°C with rocking or occasional shaking.

Add RNAse A and incubate at 37°C for 0.5-2 hours. Add 5M NaCl to the sample and vortex for 15 seconds. Centrifuge the sample at full speed for 10 minutes. Precipitate DNA from supernatant using isopropanol, followed by centrifugation. Wash with ethanol and dry DNA pellet. Resuspend in DEPC H₂O and incubate at 65⁰C for 10 minutes. Dilute extracted genomic DNA to lOOng/μl (~3xlO⁴ copies) by di-H₂O.

2) Amplification Step: polymerase chain reaction (PCR) - one or two step PCR reaction.

Primers used in both two-round (nested PCR) and one-round PCR amplification schemes to amplify exon 2 of the K-ras gene containing the area of interest (codon 12) are shown in Table 1 above. Forward primer 3 (SEQ ID NO:7) is biotinylated at the 5' end. All two-round (nested) amplification is carried out using primers 1 (SEQ ID NO: 9) and 2 (SEQ ID NO: 10) for first round amplification. Second round amplification is completed using primer 3 (SEQ ID NO:7) and either primer 2 (SEQ ID NO: 10) or 6 (SEQ ID NO: 11) to produce amplicons of different lengths. One-step PCR is carried out using primers 3 (SEQ ID NO:7) and 7 (SEQ ID NO:8).

The reaction conditions for two-round and one-round PCR are shown in Tables 3 and 4.

Table 4. Two-step PCR reaction for Ras codon 12 Table 4A. First round PCR:

Table 4B. Second round PCR:

3) PCR clean-up:

Clean-up PCR products according to the manufacturer instructions for the spin column (i.e., Qiagen).

4) Isolation of Single-Stranded Polynucleotide: Bead preparation : - Resuspend the beads in the original vial by rotation

Calculate the amount of beads required: 6μl/probe per sample x (probe number + 1), e.g., 5 probes for codon 12: (wt, 12V, 12C, 12D, 12R) requires 36μl beads.

Wash beads twice with Ix BW buffer 500μl. Bead Washing: - Place the tube containing the beads on a magnet for 1 min.

Remove the supernatant by aspiration with a pipette while the tube is on the magnet.

Remove the tube from the magnet. Add 500μl washing buffer and resuspend the beads. - Repeat Bead washing steps twice.

Single-Strand Isolation :

Resuspend beads in 2xBW buffer to twice original volume.

Add equal volume of the biotinylated PCR amplicon to the washed beads

Incubate for 15 min. at room temperature with gentle mixture no less than three times.

Place the tube in magnet for 1 minute and discard the supernatant. Wash 2 times with 500μl IxBW buffer Wash 1 time with 500μl IxSSC buffer DNA denaturing: Resuspend the binding beads with 500 μl 0.15M NaOH

Incubate for 10 min. at room temperature with gentle mixture no less than three times.

Place the tube in the magnet for 1 minute and discard the supernatant. - Wash 2 times with 500μl BW buffer as above

Wash 1 time with 500 μl EB buffer as above. Aspirate all supernatant.

5) Probe Hybridization Step:

Add 5 μl different probes (10 μM each) into 96-well plate, one probe per well.

Resuspend denatured DNA-bead mixture with 90 μl fresh-made melting buffer (5 probes per sample as example).

Take 15 μl into wells pre-loaded with probes for a total reaction volume of 20 μl..

Seal the plate. Spin the plate at lOOOrpm, 1 min. to combine the reagents and eliminate bubbles in the wells.

6) Melting Curve Generation: Melting Curves were generated on the Applied Biosystems 7500Fast Real Time

PCR instrument, using the "DISSOCIATION" assay format on the Sequence Detection Systems Software vl.4. Fluorescence filters were designated based on the reporter dye in use (SYBR Green or SYTO9). Fluorescence data was collected from 40-90⁰C at a ramp rate of 0.1°C/sec. Reported T_m values were determined by the aforementioned software. 7) Perform data analysis and detect target sequences.

Using the amplification tools and methods described herein, the ability to detect rare mutations in a sample of DNA is greatly enhanced. The amplification methods and tools (primers, PNA) described above and elsewhere herein can also be utilized to improve the detection sensitivity and efficiency in any genotyping method in which amplification is used, including a sequencing method. Example 2

The following example demonstrates the use of the method of the invention using synthetic single-stranded DNA templates.

Initial experiments using the melting curve analysis steps of the genotyping method of the invention were carried out using five 70bp segments of synthetic, single- stranded DNA corresponding to five different ras genotypes at codon 12 of the ras gene as templates, and five corresponding hybridization probes for identification of each genotype. The 70bp template strand represents the sense strand and the 15bp probe corresponds to the anti-sense strand, with respect to the ras gene. Table 5 provides the sequences of each of the ssDNA templates and probes. Underlined sequences show the position of codon 12.

Melting curves were generated by combining synthetic ssDNA templates shown in Table 5 at a concentration of 0.2μM with 0.5 μM probe (also in Table 5) and 0.5x SYBR®

Green dye (Invitrogen) in 0.5x SSC buffer. A dissociation reaction was carried out from

4O⁰C to 9O⁰C on the ABI 7500Fast real time PCR instrument (Applied Biosystems Inc.).

Referring to Figs. IA- IE, melting curves were plotted for each ssDNA template hybridized to each of the five probes (Fig. IA- wt template, Fig. IB- G12V template, Fig. 1C- G12C template, Fig. 1D-G12D template, Fig. IE- G12R template). Matched template and probe combinations (perfect hybrids) can be clearly identified from the unmatched curves (mismatched template and probe combinations) and in each case, the perfect hybrids have the highest Tm values.

Table 6 shows the actual melting temperatures (Tm values) as determined by ABI SDS vl.4 software (ABI) for each curve shown in Figs. 1A-1E. Table 6 clearly shows that matched templates and probes (e.g., wild type template with wild type probe, or a perfect hybrid) have the highest Tm when compared with those of unmatched template and probe, demonstrating the ability of the present invention to discriminate between matched and unmatched hybrids, even when the unmatched hybrids differ by only a single nucleotide. Reactions containing oligonucleotide probe or template alone show negligible background signal. Table 6. T_ms For ssDNA-Probe Hybrids

In order to demonstrate the ability to use different double-stranded DNA binding dyes in the method of the invention, the same experiment as described above and illustrated in Fig. 1 and Table 6 was repeated, but using Ix SYTO® 9 dye in the place of SYBR® Green. In this experiment, the data is shown for all ssDNA templates and probes in a single graph (Fig. 2). As shown in Fig. 2, dissociation peaks corresponding to the Tm of matched template and probe combinations are still easily identified by their significantly higher Tm values as compared to unmatched template and probe combination. Example 3

The following example demonstrates the optimization of the length of amplification products for use in the method of the invention. In this experiment, the length of amplicons (amplification products) produced from a nested PCR approach (2 -round PCR) were compared using genomic DNA isolated from tumor tissue samples from two patients diagnosed with a pancreatic adenocarcinoma. Each patient was known to have a tumor carrying a mutation in the ras gene at codon 12 (by prior sequence analysis), one resulting in a G12R mutation in Ras and one resulting in a G 12V mutation.

Briefly, DNA extraction and 2-round PCR was generally performed as described in the protocol Example 1 , using the primers described in Table 1 for 2-round PCR. In both cases, the first round of PCR (using primers 1 (SEQ ID NO:9) and 2 (SEQ ID NO: 10) generated a 300bp fragment of the K-ras gene spanning codon 12 (the selected site for analysis). A second round of PCR was performed to generate an 82bp amplification product (using primers 3 (SEQ ID NO:7) and 6 (SEQ ID NO:11)) or a 208bp amplification product (using primers 3 (SEQ ID NO:7) and 2 (SEQ ID NO: 10)). Primer 3 was biotinylated. Peptide nucleic acid (PNA) matched to the wild type sequence of ras (Table 1, SEQ ID NO: 17) was added in both cases during the first round of PCR to block the amplification of wild-type ras sequence, and to thereby augment the mutant signal. Single-stranded templates were prepared from each of the amplification products, and the 82bp and 208bp templates were compared in a melting curve analysis using the hybridization probes described in Example 1, Table 2 and using SYBR® Green as the label.

Results are shown in Figs. 3A-3D and in Tables 7 and 8 below. Figs. 3A (208 bp amplicon) and 3B (82 bp amplicon) show the melting curves for Sample #200005, which is from the patient known to have a G12R mutation. Table 7 shows the Tms calculated from the data shown in Figs. 3A and 3B. Figs. 3C (208 bp amplicon) and 3D (82 bp amplicon) show the melting curves for Sample #070060, which is from the patient known to have a G 12V mutation. Table 8 shows the Tms calculated from the data shown in Figs. 3C and 3D.

As shown in the figures and the corresponding Tables, in each patient sample, the shorter amplicon (82 bp) appears to give larger separation of matched (perfect hybrid) and unmatched (imperfect hybrid) curves, producing Tm values above 6O⁰C for the matched mutant genotype. Without being bound by theory, the inventors believe that this may be a result of improved PCR amplification of the template when a shorter amplicon is used. In this experiment, the wild-type signal from non-mutated DNA in the tumor samples was not completely inhibited, which is reflected in the observed positive wild-type melting curves in each case. This signal can be expected, since no PNA was added in the second round of PCR to inhibit amplification of the wild-type template. The melting curves for the wild-type and the matched mutant probes (G12R in Figs. 3A and 3B, G12V in Figs. 3C and 3D) appear to show two points of inflection or two peaks. The observation of these two peaks suggests that two species are present in the reaction: one that is matched to the probe, and one that is unmatched to the probe. The two peaks represent the Tm values for the matched probe and template combination (the peak with the highest Tm) and the unmatched probe and template combination (the peak with the lower Tm), and can be interpreted alone to suggest the presence of two (or theoretically more) genotypes of the target sequence to be present in the reaction. The utility of this phenomenon is discussed in Example 12. Table 7. Tm Values

Example 4

The following example demonstrates that the generation of single-stranded polynucleotide template is a critical step of the present method.

To determine whether the generation of single-stranded polynucleotide template (e.g., by removal of the anti-sense strand) is required to get sufficient probe-template hybridization for producing and interpreting melting curves, nested PCR was carried out on genomic DNA from a tissue sample of pancreatic adenocarcinoma. The amplification step was conducted generally as described in Example 1 above to produce an amplification product.

In one case, the resulting double-stranded, biotinylated PCR amplification product was purified, bound to magnetic beads, and used directly in the melting curve reaction in the presence of excess wild-type probe. In another case, prior to hybridization and melting curve analysis, a denaturation step was added. Briefly, after purification of the PCR product and binding of the product to the magnetic beads, the product was denatured by incubation in 0.15M NaOH for 10 minutes at room temperature. After the incubation, the single-stranded polynucleotide bound to the beads was washed to remove the dissociated strand, and the melting curve analysis was performed in the presence of excess wild-type probe. Figure 4A shows that the association between the template and its antisense strand (double-stranded polynucleotide) generates a much higher fluorescent signal (peak to the right) than the association between the probe and template. A closer view given in Figure 4B shows that omitting the denaturation step also gives less pronounced melting curves for the template-probe hybrid. Therefore, a denaturation step that creates a single-stranded polynucleotide template is necessary for accurate genotyping using the method of the invention. Example 5

The following Example describes the optimization of magnetic bead concentration in the melting curve reaction.

To determine the optimal amount of magnetic beads used to isolate single-stranded polynucleotides for the melting curve reactions, 200ng of double-stranded, biotinylated DNA generated from nested PCR of genomic DNA was bound to differing volumes of DynaBeads® . The nested PCR was performed generally as described in Example 1. The dsDNA was denatured with NaOH to produce single-stranded polynucleotide template, and then used in the melting curve reaction as described in Examples 1 or 4 above. Fig. 5A shows the plot of the raw fluorescence obtained for each volume of magnetic beads used. Fig. 5B shows the derivative plot of the data in 5 A. The results show that a volume of 6μl DynaBeads® gave the strongest signal, and was therefore considered the optimum volume. This example illustrates how one can optimize the method of the invention to improve the resolution and sensitivity of the assay. Example 6

The following example demonstrates the effect of the concentration of the hybridization label on Tm values in the method of the invention. In this experiment, genomic DNA was amplified by nested PCR in presence of

PNA to generate a biotinylated 208bp amplicon corresponding to the K-ras gene, using the methods generally described in Example 1 above. The double-stranded DNA was combined with different amounts of SYTO® 9 dye (Invitrogen) in the presence of 0.5X SSC buffer. Melting curves were generated from 4O⁰C to 9O⁰C using the 7500 Fast Real Time PCR System from Applied Biosystems. Fig. 6A shows the raw fluorescence measured at each dye concentration, while Fig. 6B shows the derivative of the raw data (the melting curves). Melting temperature was slightly affected by the dye concentration. The results indicate that conditions (namely concentration) can be easily optimized for different dyes to be used in the assay. Example 7

The following example compares different PCR clean-up methods on the results of melting curve analysis. Biotinylated, dsDNA (208bp) was generated from nested PCR of genomic DNA originating from a pancreatic adenocarcinoma, using the methods generally described in Example 1. In this experiment, prior to isolation of single-stranded polynucleotide and the melting curve analysis, alternate methods were used to rid the final PCR reactions from excess biotinylated primer and other PCR components. Fig. 7A shows the resulting melting curves when gel extraction purification was used (~lhour). Fig. 7B shows the resulting melting curves when the reaction was run over a spin purification column that specifically excludes oligonucleotides of less than 40bp (5min). Both methods provided clean results indicating a mutant genotype of G12D for the sample, which illustrates the tolerance of the method of the invention for multiple different clean-up techniques for the amplification product. The genotype for this sample was confirmed by direct sequencing (data not shown). Example 8

The following example shows that the addition of PNA during the amplification step of the method of the invention inhibits amplification of wild-type template and augments the signal of the mutant template.

Genomic DNA isolated from three different samples of pancreatic adenocarcinoma was subjected to nested PCR to generate a 208bp, biotinylated product using the method as generally described in Example 1. One sample was known to contain wild type ras at codon 12 (Figs. 8E and 8F and Table 11), one sample was known to contain a G12C mutant ras (Figs. 8 A and 8B and Table 9), and one sample was known to contain a G12D mutant ras (Figs. 8C and 8D and Table 10). A 15bp peptide nucleic acid (PNA) probe complementary to wild type (G 12) K-ras at codon 12 (SEQ ID NO: 17) was included in the first round of PCR for all cases. The PNA probe was either included or excluded during the second round of PCR amplification. The single-stranded polynucleotide isolation and the probe hybridization and melting curve analysis were performed generally as described in Example 1.

The results show that identification of the correct mutant signal can be accomplished using PNA in only the first round of PCR or in both rounds of PCR. Therefore, PNA can be added to one or both amplification steps. However, the addition of PNA in the second round of PCR did amplify the mutant signal in the cases where a mutation is present, which is illustrated both by the curves themselves and the Tm values assigned to the mutant curves (Figure 8B and 8D). The addition of PNA, even in both steps, still maintains the ability to accurately genotype samples containing no mutant DNA, as is illustrated by its use in the amplification of a wild type sample in Figure 8E. Accordingly, the use of PNA can be adjusted, as needed, to inhibit wild-type signal in amplification steps of the method. Table 9. Tm Values Table 10. Tm Values

Table 11. Tm Values

Example 9

The following example compares the genotyping method of the invention with direct sequencing using tissue samples of pancreatic adenocarcinomas.

To compare the accuracy of the method of the invention to direct sequencing, which is considered to be the "gold standard" for genotyping, the following experiment was performed. Using the method generally described in Example 1, genomic DNA isolated from five different formalin-fixed paraffin (FFPE) tissue samples of pancreatic adenocarcinoma (genotype unknown) was subjected to two rounds of PCR, resulting in a biotinylated product. PNA was used to inhibit wild-type amplification during the first round of PCR. The double-stranded amplification product was coupled to streptavidin- coated magnetic beads, denatured using NaOH, and the resulting single-stranded polynucleotide templates were hybridized with oligonucleotide probes for ras codon 12 (see Example 1, Table 2) in the presence of 0.5X SSC and 0.5X SYBR® Green dye, and then analyzed by melting curve analysis, all as generally described in Example 1. Aliquots of the PCR products generated in the amplification step had previously been sent for dual strand direct sequencing. Genotypes were called from the melting curve analysis in a blinded fashion, prior to review of the sequencing results.

The melting curves for each of the patient samples are shown in Figs. 9A-9E. The results showed that for each tumor sample, one of the five melting curves corresponding to a particular probe had a significantly higher Tm than the others, which allowed the determination of the genotype for each sample (Fig. 9A: wild-type; Fig. 9B: G 12V; Fig. 9C: G12C; Fig. 9D: G12C; and Fig. 9E: G12R). Figs. 9A-9E show the precise melting temperature for each curve as determined by Applied Biosystems SDS vl.4 software. The genotyping results were confirmed by dual strand direct sequencing and demonstrate 100% accuracy of the method. Abbreviated chromatograms for the forward strand are shown for each sample in Figs. 9A-9E. In total, the results demonstrate that the melting curves corresponding to the genotype of the sample (perfect hybrids) have a higher Tm than curves representing a mismatch between the probe and template. Secondary peaks seen at a higher temperature (~80°C) represent annealing of the single-stranded template with remnants of its complementary strand that was not completely removed during the denaturation step.

This experiment demonstrates that the genotyping method of the invention can be used to detect tumor genotypes with the same accuracy as direct sequencing. However, the method of the invention is much more rapid than the direct sequencing, as the latter results were available approximately two days after those of the method of the invention. Example 10

The following example further demonstrates the use of the genotyping method of the invention to genotype patient samples with the same accuracy as direct sequencing.

In this experiment, an additional thirty-two samples of pancreatic adenocarcinoma (ras genotype unknown to the method user) were genotyped using the method of the invention, and compared with direct sequencing results for the same samples. Briefly, as described in Example 9, genomic DNA isolated from FFPE samples of pancreatic adenocarcinomas was subjected to two rounds of PCR resulting in an 82bp biotinylated product. The double stranded DNA was coupled to streptavidin-coated magnetic beads, denatured using NaOH, and then analyzed by melting curve analysis. In this experiment, a Tm of greater than 6O⁰C was considered positive for any given probe.

Genotyping results were confirmed by dual strand, direct sequencing and demonstrate 100% accuracy of the method of the invention (chromatograms not shown). The results are shown in Table 12.

Table 12. Tm versus Direct Sequencing in 32 Tumor Samples

Example 11

The following example demonstrates the use of the method of the invention to detect double mutations at codon 12 of K-ras.

Using the novel genotyping method of the invention and two-step PCR amplification as generally described in Example 1 and in Examples 9 and 10 above, two different mutations at codon 12 of K-ras (G 12V and G 12D) were detected in two different tumor samples. The melting curves are shown in Figs. 1OA and 1OB (Fig. 1OA: sample 070035; Fig. 1OB: sample 070070). These results were later confirmed to be present by direct sequencing. Both of the mutant genotypes present in each sample had Tm values of greater than 6O⁰C when combined with a matched probe (to form a perfect hybrid), and therefore, the samples met the assay criteria to be positive for two mutations. Example 12

The following example demonstrates the use of the genotyping method of the invention to detect the presence of novel mutations that are not target sequences of the method.

In this experiment, genomic DNA from a tissue sample of a pancreatic adenocarcinoma was subjected to the protocol described in Examples 1 or 9-11. The sample was not positive for any of the mutations at K-ras codon 12 that were screened for in the assay (see Table 2 for probes used). However, examination of the melting curves generated show a distinct second peak when the template is combined with a wild type probe, suggesting the presence of a species in the mixture that is not perfectly complementary to the wild-type probe. Direct sequencing of the sample confirms the presence of a silent mutation at codon 11. Therefore, the method of the invention also screens for mutations not addressed in the design of the specific probes, and allows the user to identify rare, novel, or unexpected genotypes that exist in the area(s) (selected sites) covered by the probes. Example 13

The following example demonstrates the sensitivity of the method of the invention. To test the sensitivity of the method of the invention using one-step PCR, genomic

DNA extracted from the SW480 cell line, which originates from a colorectal adenocarcinoma and harbors a homozygous G 12V mutation in the K-ras gene, was combined in different ratios with human genomic fetal heart DNA (known to be wild-type in codon 12 of K-ras) for a total DNA input of 500ng per reaction. One-round PCR in the presence of PNA was used to amplify the templates, and the remainder of the procedure was completed as described in Example 1.

The results are shown in Fig. 12 and confirm that the method was able to detect the G 12V mutation at a concentration of 0.05% (0.25ng or 75 copies of mutant template) in a wild type background (a T_m of greater than 60⁰C was considered positive for a given probe).

Example 14

The following example demonstrates the use of one-step PCR in the method of the invention to genotype clinical samples.

The genotyping method of the invention as generally described in Example 1 was carried out on genomic DNA isolated from three tissue samples of pancreatic adenocarcinomas. Each initial DNA sample was divided into two aliquots and in one reaction, a one-step PCR was used to produce the amplification product, and in the other reaction, a two-step PCR was used to produce an amplification product. PNA was used to inhibit wild-type signal in both reactions. The remainder of the method was the same for each reaction. The results are shown in Figs. 13A-13F. Figs. 13A (2-step PCR) and 13B (1-step PCR) provide the results for sample 200090; Figs. 13C (2-step PCR) and 13D (1- step PCR) provide the results for sample 2000089; Figs. 13E (2-step PCR) and 13F (1-step PCR) provide the results for sample 2000060. Tables 13-15 provide the Tm values corresponding to those illustrated by the figures.

This experiment demonstrates that the use of one-step PCR gives the same genotyping results as two-step PCR for all three samples tested. Therefore, using one-step PCR is an effective and time saving alternative to the practice of the genotyping method of the invention.

Table 13. Comparison of Tm for Sample #200090

Table 15. Com arison of Tm for Sam le #200060

Example 15 The following example demonstrates altering the design of the PNA clamp (PNA wild-type block) used in the amplification step of a genotyping method in order to improve the amplification step and the sensitivity of the method.

Table 16 shows the sequences of five different PNA molecules that were designed complementary to the wild type sequence at codon 61 of K-ras. Each of the five PNA molecules differs in length and/or purine content. The ability of each PNA sequence to block the wild type signal and therefore to affect the sensitivity of the method was evaluated by mixing differing amounts of genomic DNA isolated from the cell line SW948, harboring a heterozygous Q61L mutation, with human fetal heart genomic DNA, which is known to contain only wild-type K-ras. These DNA mixtures underwent one-step PCR and melting curve analysis as generally described in Example 1. Primers OLuI 37 and OLul38 were used when testing PNA61B, PNA61C, and PNA61D (Table 17). Alternative primers, OLu 146 and OLuI 50 were used when evaluating PNA61F and were designed such that the primer annealing site overlaps by two nucleotides with the PNA binding site. The results of the example are shown in Table 18 and in Figs. 14A-14D. Increased purine content in the PNA increases the sensitivity of the method, based on the observation that PNA61C and D yield stronger signals/steeper melting curves at the mutant Tm than does PNA61B. However, it is noted that PNA61C and PNA61D anneal to antisense, whereas PNA61B anneals to sense, and so strand placement per se may also contribute to this effect. Increased length of the PNA clamp does not necessarily contribute to a higher sensitivity based on the finding that PNA61B is the longest of the 4 PNAs tested, yet exhibits the poorest sensitivity. Greatest sensitivity was observed using a PNA design with high purine content and using primers that overlap the PNA binding region. It is noted that although this example illustrates the use of PNA and primers in the amplification step of a particular genotyping method, the PNA and primers described in this example may also be used to amplify nucleic acid molecules for any other purpose, including as a nucleic acid amplification step in any other genotyping method or kit.

Table 16

Table 17

Table 18

Example 16

The following example demonstrates the use of differing probe lengths to obtain optimal discrimination between matched and unmatched nucleic acid template and probe in the genotyping method of the invention.

To utilize the genotyping method for detecting mutations in codon 61 of the K-ras gene, probes were designed that detected five different K-ras codon 61 genotypes: Q61

(wt), 61Hl, 61H2, 6 IL, and 6 IR. The probes were designed at two different lengths, 15bp and 18bp to determine which length gave optimal discrimination between templates that are matched or unmatched to the probe. Dissociation analysis in the presence of SYBR Green dye was used to determine the Tm for each probe with a synthetic single-stranded template of each of the five above mentioned genotypes. Thus, each probe should show a match (high Tm) for only one of the five synthetic DNA templates tested. The results for this example are shown in Figs. 15A-15C. The melting curves shown in Fig. 15A represent the 15bp oligonucleotide probes, and do not show a difference in Tm between matched and unmatched probe/template combinations. Fig. 15B shows the melting curves generated for the 18bp oligonucleotide probes, which do give an obvious shift in the Tm between matched vs. unmatched probe/template combinations (circled). Among the panel of probes, the 18 mer corresponding to the Q61L mutation gave the lowest Tm when hybridized to the matching synthetic ssDNA template. A 19bp probe was therefore designed and tested with a goal to increase the Tm to a value closer to that of the other matched probe/template combinations and thereby achieve comparable Tm values for all perfect matches. The results of this test are shown in Fig. 15C and demonstrate that increasing the length of the 6 IL probe by 1 nucleotide increased the Tm by 1.5°C, achieving the desired effect. Thus, adjustments in probe length can be used to fine tune Tm values to bring them into compliance with the use of universal Tm cut-offs values. Example 17 The following example demonstrates the use of the genotyping method of the invention to determine genotypes at codon 61 of the K-ras gene.

One-step PCR was carried out on four samples of genomic DNA isolated from formalin fixed paraffin embedded (FFPE) tissue samples of pancreatic adenocarcinomas that had been previously genotyped by bi-directional sequencing, each having a different genotype at codon 61 of K-ras. Amplification was accomplished using primers OLuI 37 and OLul38, and PNA61C to block the wild type signal present in the samples (See Example 15). The method described in Example 1 was used to obtain melting curves for each PCR product when tested with probes corresponding to five different genotypes at codon 61. The sequences of probes used are given in Table 19. Table 19

Results of this analysis are shown in Fig 16A-16D. Genotypes for each of the samples determined by the method were consistent with results from earlier bi-directional sequencing (data not shown), although the operator was blinded to these results at the time of analysis using the genotyping method.

The sensitivity of this design of the method was tested by titrating different amounts of genomic DNA from the cell line SW948, which harbors a heterozygous Q61L mutation in K-ras, into human fetal heart genomic DNA, known to contain only wild type

K-ras. The DNA mixtures were amplified using one-step PCR analysis and melting curves were generated using the method described in Example 1.

The results for the sensitivity test for the detection of mutation at codon 61 of K- ras are illustrated in Fig. 17. The results demonstrate this design of the method to be sensitive to detecting a mutation in a mixture containing 0.5% DNA mutant at codon 61 of the K-ras gene. In this example, the sensitivity cutoff is defined as the lowest titration where the Tm of the mutant probe is greater than 60⁰C. Example 18

The following example demonstrates the use of the genotyping method of the invention to determine genotypes at codon 600 of the BRAF gene.

Primers, probes, and a PNA clamp were designed to detect the most common mutation in the BRAF gene, V600E mutation. Sequences for the designed components are shown n Table 20. The efficacy of the design was tested using genomic DNA isolated from the cell line Colo205, reported to harbor the heterozygous V600E mutation (Davies et al, Nature. 2002 Jun 27;417(6892):949-54). One-round PCR amplification and melting curve analysis were carried out substantially as described in Example 1. The results of the test are shown in Fig 18, and demonstrate the correct genotyping call for the cell line.

To test the sensitivity of the design, differing amounts of genomic DNA from the Colo205 cell line were titrated into genomic human fetal heart DNA, known to be wild type in the BRAF gene. Again, one step PCR amplification and melting curve analysis were performed substantially as described in Example 1.

The results of the sensitivity test for BRAF V600E mutation detection are shown in Fig 19. The results demonstrate that this design of the method can detect up to 0.05% V600E mutant DNA in a wild type background. Table 20

Using the amplification tools and methods described herein, the ability to detect rare mutations in a sample of DNA is greatly enhanced. These amplification methods and tools (primers, PNA) can be utilized to improve the detection sensitivity and efficiency in any genotyping or sequencing method. Example 19

The following example demonstrates the use of the method to detect deletions in exon 19 of the EGFR gene.

Primers, a PNA clamp, and detection probes were designed to detect the most common deletion in exon 19 of the EGFR gene, consisting of an in- frame deletion from amino acids E746-A50. This in-frame deletion can occur by the deletion of nucleotides 2235-2249 or of nucleotides 2236-2250 of the coding sequence of the EGFR gene (the wild-type egfr sequence denoted herein as NM_005228). Thus, two deletion probes were designed to discriminate between the two possible genotypes. Sequences of the primers, PNA, and probes can be found in Table 21 below. The design utilizes the overlapping primer/PNA strategy that was determined to yield high sensitivity in Example 15. Genomic DNA was isolated from two human cell lines: HCC827, which harbors an E746- A750 deletion, and H1975, which does not harbor a deletion in the EGFR gene. Genomic DNA was amplified using the reaction conditions in Table 22 and melting curve analysis was performed using the designated probes as substantially described in Example 1. Table 21

Table 22

The results of this analysis are shown in Figs. 20A-20B and Table 23. Fig. 2OA shows the melting curves for cell line HCC827, demonstrating a shift in Tm above 6O⁰C and a positive result for the E746-A750 deletion (type 2). Fig. 2OB shows the melting curves for cell line H 1975, demonstrating no presence of the E746-A750 deletion and substantial blocking of the wild-type signal.

Table 23

The sensitivity of this design of the method was tested by titrating HCC 827 genomic DNA, containing the E746-A750 deletion, into human fetal heart genomic DNA, known contain only wild type copies of the EGFR gene. One step PCR was carried out and melting curves were generated using the method in Example 1.

The results of the sensitivity experiment are shown in Fig. 21. The method was able to detect the presence of the mutant DNA down to 0.01% HCC827 DNA in a wild type background, a positive result being a Tm of greater than 6O⁰C. This example underscores the utility of the method to detect deletions with a high sensitivity.

Using the amplification tools and methods described herein, the ability to detect rare mutations in a sample of DNA is greatly enhanced. These amplification methods and tools (primers, PNA) can be utilized to improve the detection sensitivity and efficiency in any genotyping or sequencing method. Example 20

The following example demonstrates the use of the genotyping method of the invention to determine genotypes at codons 12 and 61 of K-ras using a multiplexed PCR approach. Many clinical samples present challenges with respect to the small amount of patient material available to use for genotyping purposes. For this reason, a multiplexing approach was utilized in order to use a smaller amount of input DNA to get genotyping data for multiple genetic loci. This multiplexing approach was used in the genotyping method of the invention, but the method and tools (primers, PNA) can be used to amplify genetic material for use in any sequencing or genotyping method. A first round of PCR amplification was carried out using lOOng of total genomic DNA sample and a mixture of primers to amplify areas surrounding both codons 12 and 61 of the K-ras gene. The reaction conditions for the first round of amplification are detailed in Table 24. A second round of PCR amplification was then performed using lμL of the first reaction as a template. A separate second round reaction was set up for each locus of interest and included PNA to block any wild type signal present. Reaction conditions for the second round PCR amplification are given in Table 25. After PCR amplification, melting curves were generated using probes for codons 12 and 61 of K-ras as substantially described in Example 1 and in Example 17. Figs. 22A and 22B illustrate the results of this use of the method for the cell line

SW948, which harbors a heterozygous Q61L mutation, demonstrating that multiplexing in a first round PCR maintains the ability of the method to give the correct genotyping readout and conserves DNA. Table 24

Table 25

To determine whether the use of a multiplexing approach alters the sensitivity of this example of the method, titrations of genomic DNA isolated from the cell lines Pane 10.05, harboring a heterozygous G12D mutation in K-ras, and from SW948, harboring a heterozygous Q61L mutation in K-ras, were added to human fetal heart genomic DNA, which is known to contain only wild type K-ras. These DNA mixtures were used in the two step multiplexed amplification scheme detailed in Table 24 and Table 25, and analyzed using melting curves to determine the limit of detection of the method in this example.

The results of these sensitivity tests are shown in Fig. 23A for the cell line Pane 10.05 and in Fig. 23B for the cell line SW948. The sensitivity of the method using the K- ras codon 12 design can detect 0.05% mutant DNA in a wild type background, and the multiplex PCR approach shows the same sensitivity as a one-step PCR approach (see Fig. 12). Similarly, the multiplexed method using the K-ras codon 61 design can detect 0.5% mutant DNA in a wild type background, consistent with the sensitivity shown in Example 17. Therefore, multiplexing the two loci conserves valuable sample material while maintaining the efficacy and sensitivity of the method.

Using the amplification tools and methods described herein, the ability to detect rare mutations in a sample of DNA is greatly enhanced. These amplification methods and tools (primers, PNA) can be utilized to improve the detection sensitivity and efficiency in any genotyping or sequencing method. Example 21

The following example demonstrates the use of the method to determine genotypes at codons 12 and 61 of K-ras, codon 600 of BRAF, and to detect deletions in exon 19 of EGFR simultaneously using a multiplexed PCR approach.

To determine whether four genetic loci could be simultaneously genotyped using one lOOng aliquot of genomic DNA sample, multiplexing experiments using primers for four genetic loci were used: K-ras exon 2, K-ras exon 3, BRAF, and EGFR exon 19. Primers were multiplexed in an initial first round reaction in the absence of PNA clamping. One microliter of the first round amplification was then used as template for second round PCR amplifications split out for each individual target loci. The second round reactions use primers that lie internally to the primers used in the first round amplification and a single biotinylated primer for each genetic target. The second round amplification also includes PNA clamping to inhibit wild type background signal present in the sample. The method was completed using genomic DNA isolated from four cell lines: Pane 10.05 (K-ras G12D mutation), SW948 (K-ras Q61L mutation), Colo205 (BRAF V600E mutation), and HCC827 (EGFR E746-A750 deletion). Table 26 shows the sequences of the external primers used in the first round multiplexed amplification. Tables 27 and 28 show the PCR amplification strategies for the first and second round amplifications.

Table 26

Table 27

Table 28

Results for this example are shown in Figs. 24A-24D (Pane 10.05), Figs. 25A-25D (SW948), Figs. 26A-26D (Colo205); and Figs. 27A-27D (HCC827). The method identified the correct mutation for each of the four cell lines tested, suggesting that the use of multiplexed PCR in the method is feasible and results in a large savings of time and sample.

Using the amplification tools and methods described herein, the ability to detect rare mutations in a sample of DNA is greatly enhanced. These amplification methods and tools (primers, PNA) can be utilized to improve the detection sensitivity and efficiency in any genotyping or sequencing method. Example 22

The following example demonstrates the use and increased sensitivity of mutation detection of PN A61C for k-ras exon 3 (codon 61) in bi-directional sequencing of a longer amplicon flanking the whole k-ras exon 3 with the PNA inhibition of wild type amplicon. This example also demonstrates the use of additional primers and PNA in an amplification strategy that can be applied to a variety of genotyping methods.

The PNA clamp bi-directional sequencing method was performed as a two round PCR regimen; the first round PCR was a multiplex PCR reaction which amplifies both K- ras exon 2 and exon 3 from limited genomic DNA obtained from FFPE tumor tissues, or from human genomic DNA isolated from cell lines, or from fresh or frozen tissues. The second round PCR was performed for exon 2 or exon 3 in the presence of Kl-PNA or K2-

PNA61C, respectively. Table 29 shows the sequences of the primers for the first round and second round PCR, and the sequences of PNA for exon 2 and exon 3. Table 29

The procedure for PNA clamp PCR for bi-directional sequencing is performed as follows: 30ng (5-40 ng) of genomic DNA was added to a pre-made master mix of external PCR which contains 6uL of 5xHF buffer, 3uL of 2 mM dNTP, IuL of lOμM KIj- ex.S, IuL of lOμM Klh-ex.AS, IuL of lOμM K2i-ex.S, 1 uL lOμM of K2i-ex.AS, 0.3uL of Phusion DNA polymerase in a total of 30 μL volume. The external PCR was run using the following program: 98⁰C 2 min[98°C 1OS - 6O⁰C 30S - 72⁰C 20S] X30 - 72°C5m-4°C. 1 μl of the external PCR product was added to the pre-made master mix for internal k-rαs exon 2 and exon 3, respectively, which contains lOuLof 5xHF buffer, 5 μL of 2mMdNTP,

0.5 μL of Phusion DNA polymerase, primers for exon 2 (2μL of lOμM T7-klb.S, 2 μL of lOμM sp6-klb.AS) and 12 μL of 10 μM Kl-PNA or primers for exon 3 (2 μL of lOμM

T7-k2i.s and 2 μL of lOμM sp6-K2b.AS) and 12 μL of lOμM K2-PNA61C in a total of 50 μL volume.

Different internal PCR programs were tested and the following program performed the best to balance the PNA blocking and efficiency of PCR, resulting in good PCR efficiency and good detection sensitivity. The internal PCR was run as follows: 98⁰C 2 min-[98°C 10S-75°C 5S-70°C 30S-60°C* 20S] X15- [98⁰C 1OS - 75⁰C 5S - 7O⁰C 30S- 60°C30S-72°C 20S] X15 - 72°C5m-4°C (^Increments of temperature of +0.5 ⁰C at the completion of each cycle). PCR products were sequenced by Sanger sequencing. The sensitivity of PNA clamped PCR was tested using DNA from two cell lines:

Pancl0.05 contains a heterozygous G12D mutation, and SW948 contains a heterozygous Q61L mutation. The DNA from each cell line was diluted with wild type human fetal heart genomic DNA to produce samples containing different percentages of mutant DNA (50%, 20%, 10%, 5%, 2%, 1% for each cell line, and an extra dilution to 0.5% for DNA from SW948). PCR amplification with and without PNA blocking was performed, and the PCR products were sequenced using bi-directional sequencing methodology. The results showed that in the absence of PNA blocking of the wild-type sequence during amplification, bi-directional sequencing using amplified DNA as produced in this example can detect DNA containing a mutation at codon 12 down in a mixture of DNA containing 5-10% mutant DNA, and can detect DNA containing a mutation at codon 61 in a mixture of DNA containing 10% mutant DNA (data not shown). In contrast, by using PNA inhibition of wild type molecules during amplification, bi-directional sequencing detects DNA containing a mutation at codon 12 in a mixture of DNA containing as little as 1-2% of mutant DNA, and detects DNA containing a mutation at codon 61 in a mixture of DNA containing as little as 0.5-1% of mutant DNA (data not shown). Accordingly, using the amplification tools and methods described herein, the ability to detect rare mutations in a sample of DNA is greatly enhanced. These amplification methods and tools (primers, PNA) can be utilized to improve the detection sensitivity and efficiency in any genotyping or sequencing method.

While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following exemplary claims.

Claims

What is claimed is:

1. An oligonucleotide primer that hybridizes to a sequence from exon 3 of ras, the primer comprising a sequence selected from the group consisting of: SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:59, and SEQ ID NO:60.

2. An oligonucleotide primer that hybridizes to a sequence from exon 2 of ras, the primer comprising a sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, and SEQ ID NO:58.

3. An oligonucleotide primer that hybridizes to a sequence from braf, the primer comprising a sequence selected from the group consisting of: SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:49, and SEQ ID NO:50.

4. An oligonucleotide primer that hybridizes to a sequence from egfr, the primer comprising a sequence selected from the group consisting of: SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:51, and SEQ ID NO:52.

5. A kit comprising any one or more of the oligonucleotide primers of any one of Claims 1 to 4.

6. A method for amplifying a target polynucleotide, comprising amplifying a target polynucleotide from a nucleic acid sample to produce an amplification product, wherein the amplifying is performed using a pair of oligonucleotide primers, at least one of such primers being an oligonucleotide primer of any one of Claims 1 to 4.

7. A PNA molecule selected from the group consisting of: SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:17, SEQ ID NO:38 and SEQ ID NO:43.

8. A kit comprising any one or more of the PNA molecules of Claim 7.

9. A method for inhibiting the amplification of a non-target sequence, comprising amplifying a target polynucleotide in the presence of a PNA of Claim 7.

10. An oligonucleotide probe selected from the group consisting of: SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46.

11. A kit comprising any one or more of the oligonucleotide probes of Claim 10.

12. A method for detecting a polynucleotide sequence comprising contacting a polynucleotide with an oligonucleotide probe of Claim 10, and detecting whether the probe hybridizes to the polynucleotide sequence.

13. A method for detecting target sequences in a nucleic acid sample, comprising: a) amplifying one or more target polynucleotides from a nucleic acid sample to produce at least one amplification product, each amplification product containing a selected site of a target polynucleotide for detection of target sequences; wherein amplification of target polynucleotides having a non-target sequence at the selected site is inhibited, thereby enhancing amplification of target polynucleotides having a target sequence at the selected site; b) isolating single-stranded polynucleotides from the amplification product; c) contacting the single stranded polynucleotides with a hybridization probe and with a label that detects hybridization of a single-stranded polynucleotide and the hybridization probe, under conditions sufficient to cause the single-stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide; wherein the hybridization probe is configured to hybridize to a nucleic acid sequence spanning the selected site of the target polynucleotide, and wherein the hybridization probe has a nucleic acid sequence at the selected site corresponding to either (i) a target sequence, or (ii) a non-target sequence; d) detecting the melting temperature (T_m) of the hybridized polynucleotide; and e) detecting target sequences at the selected site of the target polynucleotides by detecting perfectly hybridized polynucleotides, wherein perfectly hybridized polynucleotides have a higher T_m than hybridized polynucleotides formed with a probe having a nucleic acid sequence that is different from the single-stranded polynucleotide at the selected site by at least one nucleotide.

14. The method of Claim 13, wherein step (a) is performed in the presence of a nucleic acid binding moiety that binds to a non-target sequence at the selected site and thereby inhibits the amplification of a target polynucleotide having the non-target sequence at the selected site.

15. The method of Claim 13, wherein the nucleic acid binding moiety binds to a sequence that overlaps with at least a portion of a sequence of the target polynucleotide that is bound by an oligonucleotide primer used to perform step (a) of amplifying.

16. The method of Claim 14, wherein the nucleic acid binding moiety is a peptide nucleic acid (PNA).

17. The method of Claim 13, wherein step (a) is performed using polymerase chain reaction (PCR).

18. The method of Claim 17, wherein step (a) comprises a single PCR.

19. The method of Claim 17, wherein step (a) comprises a first and a second PCR.

20. The method of Claim 19, wherein amplification of the target polynucleotide comprising a non-target sequence is inhibited during the first PCR and optionally during the second PCR.

21. The method of any one of Claims 13 to 20, wherein the PCR is performed using a pair of oligonucleotide primers for amplifying the target polynucleotide, wherein either primer comprises a selection moiety for isolating single-stranded polynucleotides from the amplification product.

22. The method of Claim 21 , wherein the selection moiety is biotin.

23. The method of Claim 22, wherein step (b) comprises binding the amplification product to streptavidin immobilized on a substrate, followed by denaturing the amplification product to produce a single-stranded polynucleotide that is bound to the substrate.

24. The method of Claim 13, wherein step (b) comprises binding the amplification product to an immobilized substrate, followed by denaturing the amplification product into single-stranded polynucleotides.

25. The method of Claim 23 or Claim 24, wherein the substrate is a magnetic bead.

26. The method of Claim 23 or Claim 24, wherein the substrate does not substantially interfere with the detection of the detectable label used in step (c).

27. The method of any one of Claims 13 to 26, wherein isolating single- stranded polynucleotides from the amplification product includes exposing the amplification product to denaturing conditions conducted in 0.15M NaOH.

28. The method of any one of Claims 13 to 27, wherein the amplification product is between about 45 and about 1000 nucleotides in length.

29. The method of any one of Claims 13 to 27, wherein the amplification product is between about 45 and about 500 nucleotides in length.

30. The method of any one of Claims 13 to 27, wherein the amplification product is less than about 300 nucleotides in length.

31. The method of any one of Claims 13 to 27, wherein the amplification product is less than about 100 nucleotides in length.

32. The method of any one of Claims 13 to 27, wherein the amplification product is less than about 80 nucleotides in length.

33. The method of any one of Claims 13 to 27, wherein the amplification product is about 70 to about 80 nucleotides in length.

34. The method of any one of Claims 13 to 33, wherein the amplification product is purified from other PCR components between steps (a) and (b).

35. The method of any one of Claims 13 to 34, wherein the label in step (c) is a double stranded nucleotide binding agent.

36. The method of Claim 35, wherein the double stranded nucleotide binding agent is an intercalating agent.

37. The method of Claim 35 or Claim 36, wherein the agent is a fluorescent dye.

38. The method of any one of Claims 13 to 37, wherein step (c) comprises distributing the single-stranded polynucleotides from step (b) into two or more aliquots, wherein each aliquot is contacted with the label and one of a plurality of hybridization probes to form a hybridized polynucleotide, each hybridization probe in the plurality having a different sequence at the selected site as compared to the other probes in the plurality; and wherein each aliquot is contacted with a different hybridization probe.

39. The method of Claim 38, wherein step (d) comprises determining the T_m for each of the hybridized polynucleotides, and wherein step (e) comprises comparing the T_m from the hybridized polynucleotides in each aliquot to the T_m from the hybridized polynucleotides in the other aliquots, wherein hybridized polynucleotides having a T_m that is significantly higher than the others is identified as including a probe that is fully complementary to the single-stranded polynucleotide, thereby identifying the target sequence or lack thereof at the selected site of the target polynucleotide.

40. The method of any one of Claims 13 to 37, wherein step (c) comprises distributing the single-stranded polynucleotides from step (b) into two or more aliquots, wherein each aliquot is contacted with the label and two or more of a plurality of hybridization probes to form the hybridized polynucleotides, each hybridization probe in the plurality and in the aliquot having a different sequence at the selected site as compared to the other probes in the plurality and in the aliquot.

41. The method of any one of Claims 13 to 40, wherein step (c) is conducted in between about 0.1X and about 0.5X SSC.

42. The method of any one of Claims 13 to 41 , wherein the hybridization probe is between about 10 and about 30 nucleotides in length.

43. The method of any one of Claims 13 to 41, wherein the hybridization probe is less than about 20 nucleotides in length.

44. The method of any one of Claims 13 to 41, wherein the hybridization probe is about 15 nucleotides in length.

45. The method of any one of Claims 13 to 44, wherein the nucleic acid sample is DNA extracted from a patient biological sample.

46. The method of any one of Claims 13 to 44, wherein the nucleic acid sample is DNA extracted from a patient tumor sample.

47. The method of any one of Claims 13 to 46, wherein the nucleic acid sample used in step (a) comprises between about 20 ng and about 1 mg of patient DNA.

48. The method of any one of Claims 13 to 46, wherein the nucleic acid sample used in step (a) comprises at least about 20 ng of patient DNA.

49. The method of any one of Claims 13 to 48, wherein the single-stranded polynucleotide used in step (c) comprises between about 20 ng and about 300 ng of polynucleotide.

50. The method of any one of Claims 13 to 49, wherein the target sequence includes a substitution of at least one nucleotide at the selected site for a different nucleotide as compared to a non-target sequence.

51. The method of Claim 50, wherein the substitution is a point mutation.

52. The method of any one of Claims 13 to 49, wherein the target sequence includes a deletion of nucleotides as compared to a non-target sequence.

53. The method of any one of Claims 13 to 52, wherein the target polynucleotide is at least a portion of a ras gene.

54. The method of Claim 53, wherein the ras gene is K-ras.

55. The method of Claim 53 or Claim 54, wherein the selected site spans a codon of ras selected from the group consisting of 12, 13, 59, 61 and 76.

56. The method of any one of Claims 13 to 52, wherein the target nucleotide is at least a portion of a gene encoding BRAF.

57. The method of any one of Claims 13 to 52, wherein the target nucleotide is at least a portion of a gene encoding epidermal growth factor receptor (EGFR).

58. The method of any one of Claims 13 to 52, wherein the method detects at least two different target sequences from the same gene.

59. The method of any one of Claims 13 to 52, wherein the method detects target sequences from at least two different selected sites in the same gene.

60. The method of any one of Claims 13 to 52, wherein the method detects target sequences from at least two different genes.

61. The method of any one of Claims 13 to 52, wherein the method detects target sequences from a virus.

62. The method of any one of Claims 13 to 52, wherein the method detects target sequences from a pathogen.

63. The method of any one of Claims 13 to 52, wherein the method detects target sequences that are escape mutations from small molecule or immune system therapeutic approaches.

64. The method of any one of Claims 13 to 52, wherein the method detects target sequences from two or more genes in the same biological pathway.

65. The method of any one of Claims 13 to 64, wherein the target sequence is a mutant sequence associated with a disease or condition, and wherein the method further comprises preparing a report for a clinician or other party that identifies the mutation or lack thereof in the target polynucleotide.

66. The method of any one of Claims 13 to 64, wherein the target sequence is a mutant sequence associated with a disease or condition, and wherein the method further comprises prescribing a mutation-specific treatment to a patient carrying the mutation.

67. A method of prescribing treatment for a cancer that includes identification of a particular mutation in the DNA of a patient, comprising: a) identifying a mutation in a target polynucleotide of a patient who has cancer by reviewing a report that identifies the mutation, wherein the mutation was detected using the method of any one of Claims 13 to 65; and b) administering to the patient a therapy that is specific for the mutation identified in the report.

68. A method to manufacture a therapeutic agent that is specific for one or more mutations associated with a disease or condition, comprising: a) producing a therapeutic agent that is specific for one or more mutations associated with a disease or condition; and b) labeling packaging containing the therapeutic agent to require the use of the method of any one of Claims 13 to 65 to confirm the presence of the specific mutation or mutations in a patient in conjunction with administration of the agent to the patient.

69. A packaged medicament that is specific for one or more mutations associated with a disease or condition, comprising: a) a therapeutic agent that is specific for one or more mutations associated with a disease or condition; and b) package labeling that requires the use of the method of any one of

Claims 13 to 65 to confirm the presence of the specific mutation or mutations in a patient in conjunction with administration of the agent to the patient.

70. A kit for detecting target sequences in a nucleic acid sample, comprising: a) at least one pair of PCR primers for producing amplification products from target polynucleotides that contain a selected site; b) one or more reagents that inhibit the amplification of target polynucleotides having a non-target sequence at the selected site; c) one or more reagents for isolating single-stranded polynucleotides from the amplification product; d) a label that detects hybridization of a single-stranded polynucleotide and a hybridization probe; and e) one or more hybridization probes configured to hybridize to a nucleic acid sequence spanning the selected site of the target polynucleotide, wherein each hybridization probe has a nucleic acid sequence at the selected site corresponding either to (i) a target sequence; or (ii) a non-target sequence.

71. The kit of Claim 70, wherein at least one of the PCR primers in the pair comprises a selection moiety for isolating single-stranded polynucleotides from the amplification product.

72. The kit of Claim 71 , wherein the selection moiety is biotin.

73. The kit of Claim 72, wherein the reagent in (c) is a streptavidin-coated substrate.

74. The kit of Claim 70, wherein the reagent in (b) is a nucleic acid binding moiety that binds to a non-target sequence at the selected site and thereby inhibits the amplification of a target polynucleotide having the non-target sequence at the selected site.

75. The kit of Claim 70, wherein the reagent in (b) is a peptide nucleic acid (PNA).

76. The kit of Claim 70, wherein the label of (d) is a double stranded nucleotide binding agent.

77. The kit of Claim 76, wherein the double stranded nucleotide binding agent is an intercalating agent.

78. The kit of Claim 76 or 77, wherein the agent is a fluorescent dye.

79. The kit of Claim 70, wherein the hybridization probe is between about 10 and about 30 nucleotides in length.

80. The kit of Claim 70, wherein the hybridization probe is less than about 20 nucleotides in length.

81. The kit of Claim 70, wherein the hybridization probe is about 15 nucleotides in length.

82. The kit of Claim 70, wherein the target sequence includes a substitution of at least one nucleotide at the selected site for a different nucleotide as compared to a non- target sequence.

83. The kit of Claim 82, wherein the substitution is a point mutation.

84. The kit of Claim 70, wherein the target sequence includes a deletion of nucleotides as compared to a non-target sequence.

85. The kit of Claim 70, wherein the target polynucleotide is at least a portion of a ras gene.

86. The kit of Claim 85, wherein the ras gene is K-ras.

87. The kit of Claim 85 or Claim 86, wherein the selected site is within a codon of ras selected from the group consisting of 12, 13, 59, 61 and 76.

88. The kit of Claim 70, |wherein the target nucleotide is at least a portion of a gene encoding BRAF.

89. The kit of Claim 70, wherein the target nucleotide is at least a portion of an epidermal growth factor receptor (EGFR) gene.

90. A method to manufacture an assay kit for screening for one or more mutations associated with a disease or condition, comprising: a) manufacturing the assay kit of any one of Claims 70 to 89, wherein the assay kit screens for one or more mutations associated with a disease or condition; and b) preparing packaging for the kit that includes instructions for the use of the assay kit prior to administration of a specific therapeutic agent that targets one of the mutations that is screened for by the assay kit.

91. A method for detecting target sequences in a nucleic acid sample, comprising: a) amplifying by polymerase chain reaction (PCR) one or more target polynucleotides from a nucleic acid sample to produce at least one amplification product, each amplification product containing a selected site of a target polynucleotide for detection of target sequences; wherein the step of amplifying is performed in the presence of a peptide nucleic acid (PNA) molecule that binds to a non-target sequence at the selected site and inhibits the amplification of a target polynucleotide having the non-target sequence at the selected site, thereby enhancing amplification of target polynucleotides having a target sequence at the selected site; and wherein the PCR is performed using a pair of oligonucleotide primers for amplifying the target polynucleotide, wherein either primer comprises biotin for isolating single-stranded polynucleotides from the amplification product; b) purifying the amplification product; c) isolating the amplification product by binding the amplification product to streptavidin conjugated to an immobilized substrate; d) exposing the bound amplification product to denaturing conditions sufficient to produce a single-stranded polynucleotide bound to the substrate; e) contacting the single stranded polynucleotides with a hybridization probe and with a double-stranded nucleotide binding agent, under conditions sufficient to cause the single-stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide; wherein the hybridization probe is configured to hybridize to a nucleic acid sequence spanning the selected site of the target polynucleotide, and wherein the hybridization probe has a nucleic acid sequence at the selected site corresponding to either (i) a target sequence, or (ii) a non-target sequence; f) detecting the melting temperature (T_m) of the hybridized polynucleotide; and g) detecting target sequences at the selected site of the target polynucleotides by detecting perfectly hybridized polynucleotides, wherein perfectly hybridized polynucleotides have a higher T_m than hybridized polynucleotides formed with a probe having a nucleic acid sequence that is different from the single-stranded polynucleotide at the selected site by at least one nucleotide.

92. A method for detecting mutated ras sequences in a nucleic acid sample, comprising: a) amplifying by polymerase chain reaction (PCR) one or more fragments of a ras gene from a nucleic acid sample to produce at least one amplification product, each amplification product containing a selected site comprising a specific codon of ras for detection of target sequences; wherein the step of amplifying is performed in the presence of a peptide nucleic acid (PNA) molecule that binds to wild-type ras at the selected site and inhibits the amplification of a fragment having a wild-type ras sequence at the selected site, thereby enhancing amplification of ras fragments having a mutated ras sequence at the selected site; and wherein the PCR is performed using a pair of oligonucleotide primers for amplifying the ras fragment, wherein one primer in the pair comprises biotin, producing an amplification product having one polynucleotide strand labeled with biotin; b) purifying the amplification product from other PCR reaction material; c) isolating the amplification product by binding biotin-labeled polynucleotide to a streptavidin molecule conjugated to a magnetic bead; d) exposing the bound amplification product to denaturing conditions sufficient to separate the non-biotin-labeled strand of the amplification product from the bound, biotin-labeled strand to produce a single-stranded polynucleotide bound to the bead; e) contacting the single stranded polynucleotide with a hybridization probe and with a fluorescent double-stranded nucleotide binding agent, under conditions sufficient to cause the single-stranded polynucleotide and the hybridization probe to form a hybridized polynucleotide; wherein the hybridization probe is configured to hybridize to the complement of a nucleic acid sequence spanning the selected site of the ras gene, and wherein the hybridization probe has a nucleic acid sequence at the selected site corresponding to either (i) a mutated ras sequence, or (ii) a wild-type ras sequence; f) detecting the melting temperature (T_m) of the hybridized polynucleotide; and g) detecting mutated ras sequences at the selected site of the ras fragment by detecting perfectly hybridized polynucleotides, wherein perfectly hybridized polynucleotides have a higher T_m than hybridized polynucleotides formed with a probe having a nucleic acid sequence that is different from the single-stranded polynucleotide at the selected site by at least one nucleotide.