BR112017024702B1 - DETECTION OF TARGET NUCLEIC ACIDS AND VARIANTS - Google Patents

Abstract

DETECTION OF TARGET NUCLEIC ACIDS AND VARIANTS. These are highly sensitive and specific methods for detecting nucleic acids, and are useful, for example, for detecting rare mutations or for detecting low-abundance variants in nucleic acid sequences. The methods involve an asymmetric incremental polymerase reaction (AIPR) followed by an exponential polymerase chain reaction (PCR).

Description

FIELD OF INVENTION

[001] The present invention relates to the field of methods for detecting nucleic acids. The methods of the invention are highly sensitive and specific and are therefore, for example, useful for detecting rare mutations, or for detecting low abundance variants in nucleic acid sequences.

BACKGROUND OF THE INVENTION

[002] Detection of nucleic acids present in very low quantities and/or at low frequency is desirable for many applications. Detection of genetic mutations, for example, is important for numerous diseases, such as cystic fibrosis, sickle cell anemia, and cancers. It is increasingly recognized that exceptionally sensitive and specific methods for mutation detection are needed, in particular, for low-input samples such as circulatory tumor DNA (ctDNA) and single-cell analysis. Current conventional methods can be hampered by a number of problems, including a high requirement for input sample DNA quantity, high cost per sample, complex and laborious workflows, insufficient sensitivity and/or specificity, and inability to detect low-abundance mutant DNA sequences within a large background of normal wild-type sequence (so-called mutant allele fraction; MAF). Almost all mutation detection methods rely on DNA amplification using polymerase chain reaction (PCR) to exponentially copy target DNA regions of interest using a DNA polymerase enzyme.

[003] When the goal is to discriminate between wild-type sequence and a variant (mutant) sequence that may differ as little as a single nucleotide base, the fidelity of the DNA polymerase enzyme can become a significant limitation for discriminatory power (affecting most mutation detection performance measures). Each polymerase enzyme has some base incorporation error rate for each possible incorrect base change, typically in the range of 0.5 to 300 errors per million base pairs amplified (i.e., 5x10-7 to 3x10-4). For many applications, these single-base polymerase base incorporation errors are tolerable. For example, if large quantities of DNA are available and the MAF is moderate to high, e.g., >10 to 20%, a standard PCR may be sufficient. However, in applications involving the detection of low-abundance variants, there is a need for ultrasensitive detection methods so that a true positive mutation can be discriminated from a false positive induced by polymerase error. Current mutation detection assays have detection limits of 10–20% MAF (Sanger sequencing), 5–10% MAF (pyrosequencing), 1–5% MAF (next-generation sequencing), and 0.1% MAF (digital PCR, COLD-PCR, ultra-deep next-generation sequencing).

[004] Digital PCR is a method that partitions a PCR reaction into many smaller individual reactions such that each reaction partition contains zero to only a very few target sequence molecules. The partitioning of all molecules is random and follows a Poisson distribution. Partitioning transforms the situation from an extremely low relative abundance of a rare sequence variant among an abundance of a wild-type sequence, to a situation where most partitions have only wild-type sequence and some partitions have a very high relative abundance of the rare variant sequence to the wild-type sequence. The result is an increase in sensitivity for rare sequence variant detection by diluting the wild-type sequence within each partition. However, polymerase error is still a significant problem even for digital PCR that can lead to false positives, which negatively impacts discriminatory performance and detection limits.

[005] Different PCR-based methods for enrichment and detection of minority alleles and mutations have been described, for example, by Milbury et al., Clin Chem. 2009 Apr; 55(4): 632-640, however, none of these methods is extremely sensitive and easy to perform.

SUMMARY OF THE INVENTION

[006] Therefore, there is an unmet need for extremely sensitive methods to detect low abundance nucleic acids with a very low frequency of false positives.

[007] The inventors have discovered that in methods employing standard PCR, DNA polymerase error rates create a performance barrier to the limit of detection due to the fact that DNA is exponentially amplified to numbers in the billions of copies, errors are randomly introduced in many copies of DNA, which includes false generation of the sequence variants of interest, and these errors are copied and amplified.

[008] The maximum sensitivity achievable in a standard digital PCR assay is also limited by the fidelity of the polymerase enzyme used in the reaction. When a wild-type sequence is incorrectly copied by the polymerase, this may create a copy that carries a false mutant sequence, and such a reaction may be read as positive for the mutant target sequence. Depending on how many PCR cycles are performed and at which cycle the false mutant sequence is introduced, the signal may be indistinguishable from that of a true positive and will therefore be read as a false positive. If such a polymerase error event occurs during a late PCR cycle, true positive signals will already have gained an “edge” on potential false positive signals and there may be a possibility of distinguishing between true positives and false positives.

[009] The methods of the invention reliably provide true positive reactions with a consistent signal advantage over any potential false positive reactions.

[0010] Therefore, the invention provides a method that has the ability to compensate for the consequences of polymerase errors to increase assay performance by at least an order of magnitude. The methods of the invention achieve exceedingly high sensitivity and specificity, have a simple workflow, and are relatively inexpensive.

[0011] The present invention provides extremely sensitive methods for detecting low abundance nucleic acids with a very low frequency of false positives. The methods of the invention generally consist of an asymmetric incremental polymerase reaction (AIPR) stage using a high annealing temperature, followed by a more conventional symmetric PCR stage using a lower annealing temperature, both performed within partitioned reactions, such as droplet digital PCR.

[0012] Methods for amplifying nucleic acids using PCR-based assays using primers with different melting temperatures (Tm) are known in the art. WO2006/094360 describes, for example, a single closed-tube PCR, in which two sequential symmetric PCRs are performed. In a first round, a nucleic acid at a locus of interest is specifically amplified using tagged locus-specific primers suitable for performing exhaustive PCR. In a second round, the amplification product of the first round is then amplified using tagged primers having a lower Tm than the tagged locus-specific primers.

[0013] Due to the high sensitivity and specificity, the methods of the invention are useful for detecting low abundance nucleic acid sequences. In particular, the methods are useful for detecting rare single base variants within a highly abundant wild-type allele, and are even useful when the total sample input quantity is low, as may be the case for circulatory tumor DNA (ctDNA) in patient blood plasma.

[0014] The methods of the invention generally consist of an asymmetric incremental polymerase reaction (AIPR) stage followed by a more conventional symmetric PCR stage. In the AIPR stage generally only one strand of the target nucleic acid sequence is copied, which generates a plurality of templates for conventional symmetric PCR. Therefore, while a conventional PCR in principle is exponential amplification, AIPR in principle is linear amplification. For amplification of any given target sequence of interest, at least two primers are designed to flank the sequence of interest, such that one primer (called the “H-primer”) has a very high melting temperature (Tm) and another primer on the opposite strand and orientation has a much lower Tm (“L-primer”). The two stages (AIPR and PCR) differ in the thermocycling conditions and the primers that are functionally active during each stage.

[0015] In the AIPR stage, the target single-stranded sequence is copied by the polymerase that is primed using the H-primer oligonucleotide complementary to one end of the target sequence of interest, and only a single copy (e.g., the sequence complementary to the template) is synthesized per thermal cycling. This is typically achieved by thermal cycling to 1) a temperature to denature the DNA into single-stranded molecules; 2) to a temperature that is permissible for annealing of the H-primer to initiate monodirectional copying of the target sequence of interest by a DNA polymerase, but not permissible for annealing of the L-primer; 3) to a temperature to allow extension of the strand synthesized by a DNA polymerase, but at which the L-primer cannot yet anneale; 4) repeating steps 1 through 3 in repeated cycles as needed, with an additional complementary copy synthesized per cycle that is primed and extended from the H-primer. The synthesized copy is the Watson-Crick complement to the single-stranded sequence to which the H-primer anneals, and thus each synthesized copy does not become a template for further amplification during any thermal cycles of the AIPR stage. Several to many rounds of AIPR copying in only one direction are accomplished by cycling, as above, the thermal conditions under which only the monodirectional H-primer is able to anneal and extend to synthesize a nucleic acid strand. Thus, from each original single-stranded template and for each thermal cycle, a unique complementary target sequence is generated, so that, for example, after X number of runs of asymmetric cycling, there will be X new complementary DNA molecules at the end of the stage for every Y number of single-stranded primer template molecules (hence the total number of new complementary DNA molecules in the reaction partition will be X*Y). For example, after 64 cycles with only 1 single-stranded target template in the reaction partition, there will be the 1 single-stranded template plus 1*64=64 complementary copies in the partition. With very high probability, the vast majority of these new molecules will be an exact complementary copy of the original template molecule, since the polymerase error rate is low (0.5 to 300 errors per million base pairs amplified) and only, for example, 64 copies of several dozen to several thousand base pairs in length are synthesized each. Even in the case of a polymerase error occurring during one of these AIPR cycles in a reaction partition that contained only 1 wild-type target sequence molecule and 0 mutant target sequences to begin with, of the 64 new DNA molecules, only 1 would be mutant out of 63 non-mutants. Such an error-induced target sequence would be potentially problematic if it occurred at the exact sequence position of highest interest, but may not be problematic if it occurred at another position. In contrast, in a true-positive reaction partition that started with 1 true mutant target sequence molecule and 0 wild-type targets, there would be 64 novel mutant-containing DNA molecules that are exceedingly rare, 63 novel mutant-containing molecules, and 1 false wild-type molecule. Therefore, in a digital PCR reaction, the true-positive reaction partitions have 62 to 64 additional mutant target molecules relative to the rarely occurring partition that now contains a false-positive mutant sequence due to polymerase error. After this AIPR stage, the conventional symmetric PCR stage begins, and the true-positive reaction partitions have approximately a log2(X) cycle “advantage” in terms of molecular copies over the false-positive reaction partitions. In other words, in the above example with X=64 asymmetric cycles, the true-positive reaction partitions will have approximately a log2(64)=6 cycle advantage.

[0016] In a digital PCR system using quenched fluorescent probes complementary to the target sequence of interest, which anneal to its target sequence and whose fluorophore is cleaved and therefore not quenched by the exonuclease activity of a polymerase, the released fluorophores accumulate on the partition and increase its fluorescent signal. Conventional PCR is continued until the true positive signal is discernible and the false positive signal is still lagging behind by a number of cycles. A threshold is drawn to separate the two signals. Other methods for detecting the conventional PCR product can also be used.

[0017] Figure 1 provides an overview of the method according to one embodiment of the invention.

[0018] One way to achieve unidirectional copying of only one template strand during the asymmetric stage while avoiding copying in the opposite direction (opposite strand) is to include only a single primer during the AIPR stage and add the second primer at the beginning of the asymmetric PCR stage. Most partition-based digital PCR methods, however, do not currently allow for the addition of reagents once partitioning has occurred, e.g., it is difficult to add reagents to reaction droplets in droplet digital PCR (ddPCR).

[0019] In one embodiment, the present invention provides a method for assay design that produces primers that allow AIPR with both primers present in the reaction using primer pairs with very different melting temperatures. A high Tm primer (H-primer) is designed for one strand at the end of the target sequence of interest and a low Tm (L-primer) is designed for the other strand at the other end of the target sequence of interest. The methods also include means for detecting specific alleles. Said means may, for example, be allele-specific probes positioned on a variant base. The AIPR stage is performed at a very high temperature where the high Tm H-primer is able to anneal efficiently and the low Tm L-primer is not. The conventional symmetric PCR stage is performed at a lower temperature where the low Tm L-primer and the high Tm H-primer are able to anneal efficiently to the specific template. If the system uses allele-specific probes, then these are preferably designed so that they bind at or near the lower annealing temperature. The Tm of the primers, and therefore the activity during both stages, can be manipulated by primer length, introduction of engineered sequence mismatches into the primer, and/or by primer modifications (e.g., by the use of variant nucleotides such as blocked nucleic acid bases [ANB], or other oligonucleotide modifications such as addition of minor groove binder [MGB]). It is essential that the activity of the L-primer is suppressed during the asymmetric AIPR stage because whenever low-Tm primer binding and extension occurs, there is symmetric copying of both strands, which increases the substrate for potential polymerase errors that will then propagate during each subsequent cycle. Because of this fact, the preferred assay design in terms of avoiding false positives is one in which the difference between the asymmetric stage annealing temperature and the low Tm initiator melting temperature is high.

[0020] Therefore, it is an aspect of the invention to provide methods for detecting the presence of a target nucleic acid sequence or detecting the presence of a variant sequence in a target nucleic acid sequence in a sample comprising the steps of

[0021] a) providing a sample comprising model nucleic acids

[0022] b) providing a primer set comprising at least one pair of primers capable specifically of amplifying the target nucleic acid sequence, wherein the primer set comprises at least one H-primer and one L-primer, wherein the melting temperature of H-primer is at least 15 °C higher than the melting temperature of L-primer, and wherein the L-primer contains a sequence complementary to a fragment of the elongation product of H-primer,

[0023] c) providing a nucleic acid polymerase that has polymerase activity at an elongation temperature,

[0024] d) preparing partitioned PCR reactions, each comprising a portion of the sample, the primer set, the nucleic acid polymerase, PCR reagents and, optionally, detection reagents

[0025] e) perform an asymmetric incremental polymerase reaction (AIPR) comprising the steps of:

[0026] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[0027] ii. incubate the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer,

[0028] iii. optionally, incubate the partitioned PCR reactions at the elongation temperature,

[0029] iv. optionally, repeat steps i to iii,

[0030] v. thereby amplify only one strand of the target nucleic acid sequence

[0031] f) perform a polymerase chain reaction (PCR) comprising the steps of:

[0032] 1) incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[0033] 2) incubate the PCR at a low annealing temperature that allows both H-primer and L-primer annealing,

[0034] 3) incubate the PCR at the elongation temperature, which allows the extension of all annealed primers

[0035] 4) optionally, repeat steps II to IV,

[0036] 5) thereby amplifying both strands of the target nucleic acid sequence to obtain an ePCR product

[0037] g) detecting whether the PCR product comprises the target nucleic acid sequence or the variant sequence in the target nucleic acid sequence.

DESCRIPTION OF DRAWINGS

[0038] Figure 1 shows an overview of an IBSAFE method. Figure 1A shows a situation where the mutant template is present. During the AIPR stage, complementary copies of the mutant sequence are generated. The temperature is kept high enough so that the L-primer and probes are not annealed. In the symmetric stage, the mutant DNA is exponentially amplified. Figure 1B shows the situation where a wild-type template is present, but a polymerase error occurs. During the AIPR stage, several copies of the wild-type sequence are generated, but only one copy of the erroneous mutant sequence. The temperature is kept high enough so that the L-primer and probes are not annealed. In the symmetric stage, both the wild-type DNA and the erroneous mutant DNA are exponentially amplified, however, since there are many copies of wild-type DNA at the beginning of the exponential phase, the wild-type DNA greatly outnumbers the mutant DNA.

[0039] Figure 2 shows two specific examples of assay designs for two target sequences of interest, both within the PIK3CA oncogene: the H1047R variant located at codon 3,140 with a nucleotide change from A to G (top), and the E542K variant located at codon 1,624 with a nucleotide change from G to A (bottom).

[0040] Figure 3 shows Droplet digital PCR plots. The Figure shows the mutant DNA signal obtained from the PrimePCRTM Mutation Assay (left side) and IBSAFE assays (right side) for PIK3CA H1047R variant (top half) and PIK3CA E542K variant (bottom half). Droplets (X-axis) are indicated as dots with their fluorescent intensity (Y-axis). Note the absence of false positive droplets within the negative control wells using the IBSAFE method (outlined in the boxes in the top right corners of each plot) compared to the PrimePCRTM Mutation Assay.

[0041] Figure 4 shows an Experimental Assay Design - PIK3CA c.3140A>G (H1047R) targeting assay design with alternating versions of Primer-H (beta 1 and beta 2). Primer-H beta 1 is shorter and therefore has a lower melting temperature than Primer-H beta 2. The probes used in this assay are custom TaqMan® MGB probes (Applied Biosystems) that contain a 5’ reporter dye (FAM or HEX), a 3’ non-fluorescent quencher, and a 3’ minor groove linker attached to the quencher molecule.

[0042] Figure 5 shows Mutant (Specific) Signal Plots Demonstrating Effect of AIPR on False Positive Signal - The experimental assay design shown in Figure 4 was used, which includes beta 1 or beta 2 H-Primer along with L-Primer and mutation-specific probes, and wild-type specific probes (see Figure 4) were run with mutation-positive DNA template (not shown; all assays detected the mutation) and no AIPR wild-type template (A), with AIPR at a lower temperature (67°C) (B), and with AIPR at a higher temperature (74°) (C). Panels D, E, and F show Mutant (Specific) Signal and No False Positive Signal with the Use of Different AIPR Annealing Temperatures and Symmetric Annealing Temperatures; (D) IBSAFE assay for PIK3CA E542K mutation. In this example AIPR is performed with an annealing temperature of 75 °C and the symmetric stage with an annealing temperature of 46 °C; (E) IBSAFE assay for E545K mutation in PIK3CA. In this example AIPR is performed with an annealing temperature of 75 °C and the symmetric stage using an annealing temperature of 46 °C; (F) IBSAFE assay for NRAS Q61R mutation with AIPR using an annealing temperature of 73 °C and the symmetric stage using an annealing temperature of 48 °C.

[0043] Figure 6 shows an example of limit of detection comparison - the mutant allele frequency measured for PrimePCR™ Mutation Assay (left side) and IBSAFE assays (right side) for PIK3CA H1047R variant (top panel and bottom panel) and PIK3CA E542K variant (middle panel) are shown. The top and middle panels show results from assays comprising template DNA comprising 0; 0.01; 0.1 and 1% mutant DNA (with the remainder being wild-type), while the bottom panel shows results from assays comprising template DNA comprising 0; 0.001; 0.01; 0.1; 1 and 10% mutant DNA. The false positive signals for the PrimePCRTM Mutation Assay indicate that the result at 0.01% MAF cannot be trusted (overlap with the negative control) and therefore the lower limit of detection is 0.1%. In contrast, for IBSAFE assays, 0.01% MAF is reliable, and even at 0.001% the results are reliable. The lower limit of detection has yet to be fully tested but is likely, depending on the assay, to be considerably lower than 0.001%.

[0044] Figure 7 shows the assay design (A) and results (B) of a method according to the invention using a misaligned modified L-primer targeting the KRAS G13D mutation.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

[0045] Amplification: Amplification of a nucleic acid is the generation of copies of said nucleic acid. The term “primer pair capable of amplifying a target nucleic acid” as used herein refers to the fact that if said primer pair is added to a PCR together with the target nucleic acid, nucleotides and a nucleic acid polymerase, then said PCR will result in the production of the target nucleic acid.

[0046] Approximately: The term approximately, as used herein, refers to +/- 10%, preferably +/- 5%, for example, +/- 1%.

[0047] Denaturation temperature: The denaturalization temperature is a temperature that allows all DNA molecules in the sample to be denaturalized and/or in the PCR reactions and/or the AIPR to be denaturalized to single-stranded molecules. Preferably, the denaturalization temperature is sufficiently low to ensure that the nucleic acid polymerase is not permanently denaturalized. Typically, the denaturalization temperature is a temperature in the range of 90 to 99 °C, such as in the range of 92 to 97 °C, for example, in the range of 94 to 95 °C.

[0048] Elongation temperature: The elongation temperature is a temperature that allows enzymatic activity of the nucleic acid polymerase. Typically a nucleic acid polymerase has activity over a range of temperatures and therefore the elongation temperature can be any temperature within the range. Most nucleic acid polymerases have an optimal temperature but retain activity at temperatures other than the optimal temperature. In such cases, the elongation temperature can be any temperature at which the nucleic acid polymerase has activity even if the temperature is not the optimal temperature. The term “nucleic acid polymerase having polymerase activity at an elongation temperature” as used herein refers to the fact that the nucleic acid polymerase has the ability to catalyze synthesis of a new strand of acid complementary to the template strand at the elongation temperature. In some embodiments of the invention, the elongation temperature is close to the melting temperature of the H-primer. Therefore, a nucleic acid polymerase may be chosen which has polymerase activity at a temperature close to the melting temperature of the H-primer and/or the H-primer may be designed to have a melting temperature close to the elongation temperature. The term “close to the temperature” as used in this connection may, for example, be within +/- 5°C, such as +/- 2°C, for example, +/- 1°C of said temperature. Generally, the elongation temperature is in the range of 65 to 80°C, for example, in the range of 68 to 75°C.

[0049] Melting temperature: The melting temperature of a primer is the temperature at which 50% of the primer forms a stable double helix with its complementary sequence and the other 50% is cleaved into single-stranded molecules. The melting temperature may also be denoted as Tm. Preferably, Tm as used herein is calculated using a nearest-neighbor method based on the method described in Breslauer et al., Proc. Natl. Acad. Sci. 83, 3746-3750 (1986) using a parameter of 50 mM salt concentration and 900 nM primer concentration. For example, the method is implemented by the "Multiple Primer Analyzer" software from Life Technologies/Thermo Fisher Scientific Inc.

[0050] The term “primer pair capable of amplifying a target nucleic acid” as used herein refers to the fact that if said primer pair is added to a PCR together with the target nucleic acid, nucleotides, nucleic acid polymerase and other PCR reagents, then said PCR will result in the production of the target nucleic acid. One of the primers of the primer pair will be a forward primer, while the other will be a reverse primer. If the H-primer is a forward primer, then preferably the L-primer will be a reverse primer and vice versa.

[0051] PCR reagents: PCR reagents are reagents that are added to a PCR in addition to the nucleic acid polymerase, sample and primer set. PCR reagents comprise at least nucleotides. In addition to PCR reagents they may comprise other compounds such as salt(s) and buffer(s).

[0052] H-initiator and L-initiator: An H-initiator is an initiator that has a high melting temperature, while an L-initiator is an initiator that has a low melting temperature.

[0053] Primer set: A primer set contains two or more different primers. A primer set contains at least one pair of primers capable specifically of amplifying a target nucleic acid. Furthermore, a primer set according to the invention contains at least one H-primer and one L-primer. Therefore, in embodiments of the invention, where the primer set contains only two different primers, then the primer set contains one H-primer and one L-primer, wherein the H-primer and L-primer are capable of amplifying a target nucleic acid.

[0054] Target nucleic acid: Any nucleic acid sequence whose presence it is desirable to detect. For example, the target nucleic acid may be a nucleic acid sequence associated with a clinical condition.

METHOD FOR DETECTING A VARIANT SEQUENCE OR A TARGET NUCLEIC ACID

[0055] The present invention provides methods for detecting the presence of a variant sequence in a target nucleic acid in a sample.

[0056] Such methods may be useful for detecting whether a variant sequence is present in a sample, which may comprise a mixture of target nucleic acids in which only a portion of the target nucleic acids may comprise the variant sequence. In particular, the methods are useful for detecting the presence of a variant sequence in a sample comprising target nucleic acids of which only a small fraction may potentially comprise the variant sequence.

[0057] Said variant sequence may be any variant sequence that is desirable to detect. For example, the variant sequence may be associated with a clinical condition as described in more detail herein below in the section “Method of predicting the presence of a clinical condition”. In particular, the variant sequence may be any of the variant sequences described below in the section “Variant sequence and target nucleic acid sequences”.

[0058] The sample may be any sample in which it is desirable to detect whether said variant sequence is present. For example, if the variant sequence is indicative of a clinical condition, the sample may be a sample from an individual at risk of acquiring said clinical condition.

[0059] The present invention also provides methods for detecting the presence of a target nucleic acid sequence in a sample.

[0060] Such methods may be useful for detecting whether a target nucleic acid sequence is present in a sample. Said sample may comprise a mixture of template nucleic acids that potentially comprise the target nucleic acid sequence. In particular, the methods are useful for detecting the presence of a target nucleic acid sequence in a sample, which may potentially comprise only a very low level of said target nucleic acid sequence.

[0061] Said target nucleic acid sequence may be any target nucleic acid sequence that is desirable to detect. For example, the presence of the target nucleic acid sequence may be associated with a clinical condition as described herein below in more detail in the section “Method of predicting the presence of a clinical condition”. In particular, the target nucleic acid sequence may be any of the target nucleic acid sequences described below in the section “Variant sequence and target nucleic acid sequence”.

[0062] The sample may be any sample in which it is desirable to detect whether said target nucleic acid sequence is present. For example, if the target nucleic acid sequence is indicative of a clinical condition, the sample may be a sample from an individual at risk of acquiring said clinical condition.

[0063] Methods of detecting the presence of a target nucleic acid sequence or the presence of a variant nucleic acid in a sample generally comprise the following steps:

[0064] a) providing a sample comprising model nucleic acids;

[0065] b) providing a primer set which, for example, may be any of the primer sets described in this document below in the “Primer Set” section,

[0066] c) providing a nucleic acid polymerase that has polymerase activity at an elongation temperature, which for example, may be any of the nucleic acid polymerases described in this document below in the “PCR reagents” section;

[0067] d) preparing partitioned PCR reactions, for example, as described in this document below in the section “Partitioned PCR Reactions”

[0068] e) performing an asymmetric incremental polymerase reaction (AIPR), for example, as described in this document below in the section “Asymmetric incremental polymerase reaction”

[0069] f) perform a polymerase chain reaction (PCR), preferably an exponential PCR reaction as described in this document below in the “Exponential PCR” section

[0070] g) detecting whether the PCR product comprises the target nucleic acid sequence or the variant sequence in the target nucleic acid sequence, wherein said detection, for example, may be carried out as described herein below in the “Detection” section.

[0071] Each partitioned PCR reaction must comprise at least part of the sample, the primer set, and sufficient PCR reagents to enable a PCR reaction. Methods and reagents useful for performing a PCR reaction are well known to one of ordinary skill in the art. For example, each partitioned PCR reaction may comprise any of the nucleic acid polymerase and PCR reagents described herein below in the “PCR reagents” section.

[0072] Depending on whether the detection mode related to the PCR product comprises the variant sequence or not, each partitioned PCR reaction may also comprise detection reagents, such as any of the detection reagents described below in the “Detection” section.

[0073] The methods of the invention are useful, for example, for applications requiring high performance discrimination between any two sequences with numerous nucleotides. For example, the methods of the invention are useful for applications requiring high performance discrimination between any two sequences that differ by only one or a few nucleotide bases and where the inherent polymerase base incorporation error rate can lead to false positive target sequences of interest. The methods can be used with probe-based discrimination of single nucleotide variants, for example, as described herein below in the “Detection” section, or with primer-based discrimination. The method can be applied to any type of modified or unmodified deoxyribonucleic or ribonucleic acid (DNA/RNA) sequences of interest in any organisms and of any length from a few dozen nucleotides in length to hundreds to thousands of nucleotides in length. The method can be used with modified or unmodified primers, with or without modified or unmodified probes. The method can be performed in multiplexing with many simultaneous interrogations of multiple target sequences of interest.

[0074] In general, the methods of the invention have a very low limit of detection. This allows detection of target nucleic acid sequence potentially present at very low levels, and/or detection of the presence of variant sequences potentially present at very low levels in mixtures comprising other target nucleic acid sequences. In general, a large difference between the melting temperature of primer-H and primer-L may allow a very low limit of detection. Useful melting temperatures of primer-H and primer-L are described herein below. Also a large difference between the applied high annealing temperature and low annealing temperature may allow a very low limit of detection. Useful high and low annealing temperatures are described herein below.

[0075] The detection limit can be determined in a number of ways. For example, the detection limit can be determined by determining the minimum mutant allele fraction (MAF) that can be reliably differentiated from a negative control that contains only wild-type template. The mutant allele fraction is the fraction of mutant alleles detected compared to the total number of alleles (wild-type plus mutant) detected. In theory, the MAF should be zero when the input template is wild-type only, however, due to false positives, the fraction can be above zero. False positives lead to a poorer detection limit for a method due to the fact that very low MAF in a true positive cannot be distinguished from the very low MAF detected in a true negative. Preferably, the methods of the invention have a MAF detection limit that is less than 0.01%, e.g., it can be less than 0.001%. The MAF can, for example, be determined as described herein below in Example 1.

A KIT OF PARTS

[0076] The present invention also provides a kit of parts comprising:

[0077] a) a primer set, which for example, may be any of the primer sets described in this document below in the “Primer Set” section,

[0078] b) a detection probe is capable of hybridizing to the target nucleic acid sequence, said probe being linked to at least one fluorophore and at least one quencher, wherein said detection probe, for example, may be any of the detection probes described in this document below in the “Detection” section,

[0079] c) a nucleic acid polymerase which, for example, may be any of the nucleic acid polymerases described in this document below in the “PCR reagents” section;

[0080] d) PCR reagents which, for example, may be any of the reagents described in this document below in the “PCR reagents” section;

[0081] e) reagents for preparing droplets containing partitioned PCR reactions, which for example, may be any of the reagents described in this document below in the section “Partitioned PCR Reactions”.

[0082] Parts kits are particularly useful for carrying out the methods of the invention.

VARIANT SEQUENCE AND TARGET NUCLEIC ACID SEQUENCE

[0083] As described above, the methods of the invention are useful for detecting the presence of a variant sequence in a target nucleic acid sequence.

[0084] Often, said variant sequence may be a mutated sequence. Therefore, the target nucleic acid sequence may be a nucleic acid sequence, which may be present as a wild-type sequence or a mutated sequence.

[0085] It is also possible that the variant sequence is a polymorphism and therefore the target nucleic acid sequence may be present as several different polymorphs. In order to simplify the discussion, the most commonly occurring target nucleic acid sequence is referred to herein also as the “wild-type sequence”, although strictly speaking also the variant sequence in some circumstances may be considered a wild-type sequence.

[0086] Therefore, the methods of the invention may be methods for detecting the presence of a variant sequence in a target nucleic acid sequence, wherein said target nucleic acid sequence may potentially be present as a wild-type sequence or it may comprise the variant sequence.

[0087] The variant sequence may differ from the wild-type sequence by substitution(s), deletion(s) and/or insertion(s). It may be preferred that both the wild-type sequence and the variant sequence can be amplified in a PCR reaction by the primer pair capable specifically of amplifying the target nucleic acid sequence. Accordingly, it may be preferred that the wild-type sequence and the variant sequence do not differ greatly from each other in length. The variant sequence may, for example, differ from the wild-type sequence by insertion in the range of 1 to 1,000 nucleotides, such as in the range of 1 to 100 nucleotides, for example, in the range of 1 to 50 nucleotides, such as in the range of 1 to 10 nucleotides, for example, in the range of 1 to 5 nucleotides, such as an insertion of 1 nucleotide. Similarly, the variant sequence may, for example, differ from the wild-type sequence by a deletion in the range of 1 to 1000 nucleotides, such as in the range of 1 to 100 nucleotides, for example, in the range of 1 to 50 nucleotides, such as in the range of 1 to 10 nucleotides, for example, in the range of 1 to 5 nucleotides, such as a deletion of 1 nucleotide. The variant sequence may also differ from the wild-type sequence by a substitution, for example, by a substitution in the range of 1 to 1000 nucleotides, such as in the range of 1 to 100 nucleotides, for example, in the range of 1 to 50 nucleotides, such as in the range of 1 to 10 nucleotides, for example, in the range of 1 to 5 nucleotides, such as a substitution of 1 nucleotide.

[0088] Therefore, in one embodiment of the invention, the variant sequence may differ from the wild-type sequence by only one nucleotide, for example by the deletion, insertion or substitution of 1 nucleotide. Therefore, the variant sequence may be a single nucleotide variation or single nucleotide mutation. The variant sequence may also be a polymorphism, such as a single nucleotide polymorphism.

[0089] As explained above, it may be preferred that both the wild-type sequence and the variant sequence can be amplified in a PCR reaction using a primer pair capable specifically of amplifying the target nucleic acid sequence. Said primer pair consists of a forward primer and a reverse primer. It is preferred that the forward primer comprises or even consists of a sequence identical to a part of the target sequence that is present in both the target wild-type sequence and the target sequence comprising the variant sequence. However, it is also possible that the forward primer comprises or even consists of a sequence identical to a part of the target sequence except for some mismatches, for example up to 10 mismatches, such as up to 5 mismatches, for example up to 2 mismatches. Similarly, it is preferred that the reverse primer comprises or consists of a sequence complementary to a portion of the target sequence that is present in both the target wild-type sequence and the target sequence comprising the variant sequence. However, it is also possible that the reverse primer comprises or consists of a sequence complementary to a portion of the target sequence except for a few mismatches, e.g., except for up to 10 mismatches, such as up to 5 mismatches, e.g., up to 2 mismatches. Primers may contain mismatches for a variety of reasons, e.g., in order to adjust the primer to a suitable Tm. Thus, the methods of the invention will result in amplification of both the target wild-type sequence and the target sequence comprising the variant sequence. Therefore, the PCR product may comprise both the target nucleic acid sequence comprising the variant sequence and the target nucleic acid sequence lacking the variant sequence.

[0090] The presence of the variant sequence may then be determined by any method available to the skilled person, for example, as described herein below in the “Detection” section.

[0091] As described in this document above, the methods of the invention can be used to discriminate between two very similar sequences and therefore detect the presence of a variant sequence similar to a wild-type sequence.

[0092] There may be many different reasons why it is desirable to detect a particular variant sequence. For example, the variant sequence may be associated with a clinical condition or a risk of acquiring a clinical condition. The variant sequence may also provide a profile or at least contribute to a profile of an individual, which aids in the identification of an individual. This may have forensic applications.

[0093] However, the methods of the invention may also be used simply to detect the presence of a particular target nucleic acid sequence. Said target nucleic acid sequence may be any nucleic acid sequence that it is desirable to detect. For example, it may be desirable to detect the presence of nucleic acids from a foreign pathogen. It is generally preferred that the target nucleic acid sequence be suitable as a template for nucleic acid polymerases.

STARTER SET

[0094] The methods described herein involve the use of a primer set. The primer set comprises at least one pair of primers capable specifically of amplifying the target nucleic acid sequence, and the primer set comprises at least one H-primer and one L-primer.

[0095] It is understood within the invention that the H-primer and the L-primer may constitute a pair of primers capable specifically of amplifying the target nucleic acid sequence. Therefore, in some embodiments of the invention the primer set may consist of the H-primer and the L-primer.

[0096] In certain embodiments of the invention, partitioned PCR reactions contain only the following nucleic acids: nucleic acids present in the sample, H-primer, L-primer, free nucleotides, and optionally one or more detection probes.

[0097] However, it is also possible that the primer pair capable of specifically amplifying the target nucleic acid sequence are different from the H-primer and the L-primer. It is also understood by the invention that the L-primer together with a primer, which is not the H-primer constitutes the primer pair capable of specifically amplifying the target nucleic acid sequence.

[0098] The primer pair capable specifically for amplification of the target nucleic acid sequence consists of two primers, which may be called a forward primer and a reverse primer. The forward primer is preferably capable of annealing to the complementary strand of the target nucleic acid sequence at or near the 5' end of the target nucleic acid sequence. Preferably, the forward primer comprises a sequence identical to the 5' end of the target nucleic acid sequence. The forward primer may even consist of a sequence identical to the 5' end of the target nucleic acid sequence. In the event that the forward primer comprises a sequence not identical to the target nucleic acid sequence, it is preferable that the 3' end of the primer consists of a sequence identical to the target nucleic acid sequence. The reverse primer is preferably capable of annealing to the target nucleic acid sequence at or near the 3' end of the target nucleic acid sequence. Preferably, the reverse primer comprises a sequence complementary to the 3' end of the target nucleic acid sequence. The reverse primer may even consist of a sequence complementary to the 3' end of the target nucleic acid sequence. In the case where the reverse primer comprises a sequence non-complementary to the target nucleic acid sequence, it is preferable that the 5' end of the primer consists of a sequence complementary to the target nucleic acid sequence.

[0099] It is contemplated that the forward initiator may be an H-initiator, and the reverse initiator may be an L-initiator.

[00100] It is also contemplated that the forward initiator may be an H-initiator, and the reverse initiator may be an initiator that is neither an H-initiator nor an L-initiator.

[00101] It is also contemplated that the forward primer may be L-primer, and the reverse primer may be H-primer.

[00102] It is also contemplated that the forward initiator may be an L-initiator, and the reverse initiator may be an initiator that is neither an H-initiator nor an L-initiator.

[00103] In embodiments of the invention wherein the forward primer is primer-H, it may be preferred that the PCR reactions comprise only reverse primers that have a melting temperature that is at least 10 °C lower, preferably at least 15 °C lower, such as at least 20 °C lower, e.g. at least 25 °C lower, such as at least 30 °C, such as in the range of 15 to 50 °C, e.g. in the range of 15 to 40 °C lower than the melting temperature of primer-H. In embodiments of the invention wherein the primer set comprises a plurality of primer pairs consisting of a primer-H and a reverse primer, then each reverse primer preferably has the aforementioned melting temperature relative to the primer-H of the primer pair.

[00104] In embodiments of the invention wherein the reverse primer is primer-H, it may be preferred that the PCR reactions comprise only forward primers that have a melting temperature that is at least 10°C lower, preferably at least 15°C lower, such as at least 20°C lower, e.g. at least 25°C lower, such as at least 30°C, e.g. in the range 15 to 50°C lower, e.g. in the range 15 to 40°C lower than the melting temperature of primer-H. In embodiments of the invention wherein the primer set comprises a plurality of primer pairs consisting of a primer-H and a forward primer, then each forward primer preferably has the aforementioned melting temperature relative to the primer-H of the primer pair.

[00105] Primers may be any oligonucleotide or nucleic acid capable of acting as a DNA synthesis initiation point under suitable conditions. Such conditions may include those of AIPR or PCR described herein below in the section “Asymmetric Incremental Polymerase Chain Reaction” or “Exponential PCR”.

[00106] In some cases, an initiator may be detectably identified. In some cases, an initiator is not detectably identified.

[00107] The length of the primers may depend on the sequence of the target nucleic acid sequence. As explained elsewhere herein, the H-primer has a melting temperature that is significantly higher than the melting temperature of the L-primer. If the primer set contains more primers than the H-primer and the L-primer, then it is preferable that the remaining primers are designed to have a melting temperature similar to the melting temperature of the L-primer or lower than the melting temperature of the L-primer. However, it is understood in the invention that the primer set may comprise more than one H-primer. In such cases it is preferable that any primer, which together with any of the H-primers is capable of amplifying a target nucleic acid has a melting temperature similar to the melting temperature of the L-primer or lower than the melting temperature of the L-primer.

[00108] Therefore, it is preferred that the primer set comprises primer-H and primer-L, wherein primer-H has a melting temperature that is at least 10 °C higher than the melting temperature of all other primers in the primer set. For example, primer-H may have a melting temperature that is at least 12 °C, such as at least 14 °C, e.g., at least 16 °C, such as at least 18 °C, e.g., at least 20 °C higher than the melting temperature of all other primers in the primer set. Primer-H may have an even higher melting temperature, for example, a melting temperature that is at least 25 °C above, such as at least 30 °C above, for example, a melting temperature that is in the range of 15 to 50 °C, such as in the range of 15 to 40 °C higher than the melting temperature of all other primers in the primer set. If the partitioned PCR reactions also comprise one or more probes, such probes may also have a melting temperature that is at least 10 °C, such as at least 12 °C, such as at least 14 °C, for example, at least 16 °C, such as at least 18 °C, for example, at least 20 °C lower than the melting temperature of primer-H. However, probes may also have higher melting temperatures.

[00109] In one embodiment, primer-H is the only primer in the set of primers that has a melting temperature at least 10 °C higher than the melting temperature of primer-L. In said embodiment all other primers have a melting temperature that is at most 10 °C higher than the melting temperature of primer-L. For example, all primers except primer-H may have a melting temperature within the range of +/- 10 °C of the melting temperature of primer-L, such as within the range of +/- 8 °C of the melting temperature of primer-L, for example, within the range of +/- 6 °C of the melting temperature of primer-L, such as within the range of +/- 4 °C of the melting temperature of primer-L. If the partitioned PCR reactions also comprise one or more probes, said probes may also have a melting temperature within the range of +/- 10 °C of the L-primer melting temperature, such as within the range of +/- 8 °C of the L-primer melting temperature, for example within the range of +/- 6 °C of the L-primer melting temperature, such as within the range of +/- 4 °C of the L-primer melting temperature. However, it is also understood within the invention that the probes have a higher melting temperature, for example a melting temperature up to 20 °C higher than the L-primer melting temperature.

[00110] In one embodiment of the invention, it is preferred that the primer set does not comprise any primers:

[00111] a) having a melting temperature that is in the range of +/- 15 °C, preferably in the range of +/- 20 °C, such as in the range of +/- 25 °C, for example in the range of +/- 10 °C of the H-initiator melting temperature, such as within the range of +/- 8 °C of the H-initiator melting temperature, for example within the range of +/- 6 °C of the H-initiator melting temperature, such as within the range of +/- 4 °C of the H-initiator melting temperature; and

[00112] b) which, together with the H-primer, can constitute a pair of primers capable specifically of amplifying the target nucleic acid sequence.

[00113] In one embodiment of the invention, it is preferred that all primers within the primer set, which together with primer-H may constitute a primer pair capable specifically of amplifying the target nucleic acid sequence, have a melting temperature that is at least 10 °C, preferably at least 15 °C, such as at least 20 °C, for example at least 25 °C, such as at least 30 °C, for example in the range 15 to 50 °C, such as in the range 15 to 40 °C lower than the melting temperature of primer-H.

[00114] Therefore, it is preferred that the primer set comprises primer-H and primer-L, wherein primer-H has a melting temperature that is at least 10 °C higher than the melting temperature of all other primers in the primer set. For example, primer-H may have a melting temperature that is at least 12 °C, such as at least 14 °C, e.g., at least 16 °C, such as at least 18 °C, e.g., at least 20 °C higher than the melting temperature of all other primers in the primer set. If the partitioned PCR reactions also comprise one or more probes, such probes may also have a melting temperature which is at least 10 °C, such as at least 12 °C, such as at least 14 °C, for example, at least 16 °C, such as at least 18 °C, for example, at least 20 °C lower, for example, in the range of 20 to 45 °C lower than the H-primer melting temperature. However, it is also understood in the invention that the probes may have a higher melting temperature, even a melting temperature similar to the H-primer melting temperature.

[00115] The skilled person will be able to design primers that have an appropriate melting temperature. In general, the melting temperature of a primer may depend on the length of the primer, the sequence of the primer, and also the presence of nucleotide analogs. There are some restrictions on the sequence of the primer, due to the fact that it must be able to anneal to the target nucleic acid sequence and/or the complementary sequence. Therefore, within the restriction on the sequence, the skilled person can design a primer that has the desired melting temperature by adjusting the length of the primer. The melting temperature (Tm) can be determined as described herein above in the “Definitions” section.

[00116] The melting temperature may also depend on the presence of nucleotide analogues and therefore primers having an appropriate melting temperature may be designed by designing primers comprising one or more nucleotide analogues. The melting temperature may also depend on the presence of nucleotide mismatches in the target nucleic acid and therefore primers having an appropriate melting temperature may be designed which comprise one or more nucleotide mismatches.

[00117] Primers may incorporate additional features that allow detection or immobilization of the primer, but do not alter a basic property of the primer (e.g., acting as a DNA synthesis initiation point). For example, primers may contain an additional nucleic acid sequence at the 5' end that does not hybridize to the target nucleic acid sequence or to the sequence complementary to the target nucleic acid sequence, but that facilitates cloning or detection of an amplified product. For example, the additional sequence may comprise a restriction enzyme cleavage and/or recognition site. A region of the primer that is sufficiently complementary to a template to hybridize may be referred to herein as a hybridization region. Primers may also be linked to labels, e.g., fluorescent, functionalized, or splice labels. Said labels may be attached to their ends, sugars, or bases. Primers may also contain 3' end mismatches in designs where the primer discriminates between wild-type and variant target nucleic acid sequences, to decrease or extinguish unwanted template sequence elongation.

[00118] The primer may be single-stranded DNA prior to binding to a template nucleic acid. In some cases, the primer initially comprises double-stranded sequence, e.g., the primer may form a hairpin loop. Therefore, in general, a primer is a polynucleotide or oligonucleotide, and often primers are DNA. However, primers according to the invention may comprise one or more nucleotide analogues as well as comprising ribonucleic acid (RNA).

[00119] Nucleotide analogues are well known in the art, and the primers and probes of the invention may incorporate any useful nucleotide analogue. Nucleotide analogues may, for example, be nucleotide analogues having a modified sugar group, locked nucleic acid (LNA) nucleotide analogues, peptide nucleic acid (PNA) nucleotide analogues, glycol nucleic acid (GNA) nucleotide analogues, threosic nucleic acid (TNA) nucleotide analogues, bicyclic and tricyclic nucleoside analogues, phosphonomonoester nucleic acids incorporating a phosphorus group in the backbone, or heterocyclic, polycyclic compounds, which may be used in place of one or more of the naturally occurring heterocyclic base chemical moieties.

[00120] In another embodiment, a primer or probe used in methods and compositions described herein may comprise one or more universal nucleosides. Non-limiting examples of universal nucleosides are 5-nitroindole and inosine.

[00121] Primers can be designed according to known parameters to avoid secondary structures and self-hybridization.

[00122] Primers are commercially available from a number of suppliers and can be prepared by a variety of methods, including, but not limited to, cloning of appropriate sequences and direct chemical synthesis using methods well known in the art (Narang et al., Methods Enzymol. 68:90 (1979); Brown et al., Methods Enzymol. 68:109 (1979)).

INITIATOR-H AND INITIATOR-L

[00123] The methods and kits of the invention involve the use of a primer set comprising an H-primer and an L-primer, wherein the melting temperature of primer-H is at least 10 °C, preferably at least 15 °C higher than the melting temperature of primer-L, and wherein primer-L contains a sequence complementary to the elongation product of primer-H. The primer set may comprise other primers, for example as described herein above in the “Primer Set” section, however, the primer set may also consist of primer-H and primer-L, wherein primer-H and primer-L are capable specifically of amplifying the target nucleic acid sequence.

[00124] Primer-H is preferably designed as a primer for amplification of the target sequence or the sequence complementary to the target sequence. Therefore, primer-H preferably has the ability to anneale to both the target nucleic acid sequence and the sequence complementary to the target nucleic acid sequence. For example, primer-H may have the ability to anneale to the complementary strand of the target nucleic acid sequence at or near the 5' end of the target nucleic acid sequence, or primer-H may have the ability to anneale to the target nucleic acid sequence at or near the 3' end of the target nucleic acid sequence. Therefore, primer-H may comprise a sequence identical to the 5' end of the target nucleic acid sequence. Primer-H may even consist of a sequence identical to the 5' end of the target nucleic acid sequence. Primer-H may also comprise a sequence identical to the target nucleic acid sequence. Therefore, the H-primer may comprise a sequence complementary to the 3' end of the target nucleic acid sequence. The H-primer may even consist of a sequence complementary to the 3' end of the target nucleic acid sequence.

[00125] Similarly, primer-L is preferably designed as a primer for amplification of the target sequence or the sequence complementary to the target sequence. If primer-H is designed for amplification of the target sequence, primer-L is preferably designed for amplification of the sequence complementary to the target sequence and vice versa. Therefore, primer-L preferably has the ability to anneale to the target nucleic acid sequence or the sequence complementary to the target nucleic acid sequence. If primer-H has the ability to anneale to the target nucleic acid sequence, then primer-L preferably has the ability to anneale to the sequence complementary to the target nucleic acid sequence and vice versa. For example, the L-primer may be capable of annealing to the complementary strand of the target nucleic acid sequence at or near the 5' end of the target nucleic acid sequence, or the L-primer may be capable of annealing to the target nucleic acid sequence at or near the 3' end of the target nucleic acid sequence. Therefore, the L-primer may comprise a sequence identical to the 5' end of the target nucleic acid sequence. The L-primer may even consist of a sequence identical to the 5' end of the target nucleic acid sequence. The L-primer may also comprise a sequence identical to the target nucleic acid sequence. Therefore, the L-primer may comprise a sequence complementary to the 3' end of the target nucleic acid sequence. The L-primer may even consist of a sequence complementary to the 3' end of the target nucleic acid sequence.

[00126] In one embodiment of the invention the H-primer comprises or consists of a nucleotide sequence, which is identical to the sequence at the 5' end of the target nucleic acid sequence and the L-primer comprises or consists of a sequence identical to the complementary sequence of the 3' end of the target nucleic acid sequence.

[00127] In another embodiment of the invention, primer-L comprises or consists of a nucleotide sequence that is identical to the sequence at the 5' end of the target nucleic acid sequence and primer-H comprises or consists of a sequence identical to the sequence complementary to the 3' end of the target nucleic acid sequence.

[00128] In yet another embodiment of the invention, the L-primer consists of two parts, a part that is complementary or identical to a fragment of the target nucleic acid sequence and another part that is not complementary or identical to the target nucleic acid sequence. Such primers may also be referred to as “misaligned modified L-primer” herein. Said part that is complementary or identical to a fragment of the target nucleic acid sequence may have a very low melting temperature, and may be referred to as “non-misaligned L-primer part”. The non-misaligned L-primer part may typically consist of the range of 7 to 15 nucleotides, for example, in the range of 7 to 12 nucleotides. Said part that is not complementary or identical to the target nucleic acid sequence may contain a random sequence and may be referred to as “misaligned L-primer part”. The misaligned part of primer-L may typically consist of 2 to 8 nucleotides, such as in the range of 2 to 6 nucleotides, but could be 1 nucleotide or >8 nucleotides. This may in particular be the case in embodiments of the invention comprising a low temperature PCR step as described herein above in the section “Low temperature PCR”. The 3' end of said primer-L may for example be at a base ranging between a true target nucleic acid sequence and a non-target near-homologous one. The misaligned part, upon incorporation into newly synthesized nucleic acids and subsequent synthesis of the complementary 2nd strand, may become a suitable hybridization region.

[00129] Primer-H and Primer-L are designed to have melting temperatures as indicated herein. The skilled person will be able to design Primer-H and Primer-L to have the desired melting temperature by adjusting the primer sequence, primer length, and optionally incorporating nucleotide analogs as described herein above in the “Primer Set” section.

[00130] The H-primer is designed such that the H-primer has an annealing temperature that is significantly higher than the annealing temperature of the L-primer, e.g., at least 10 °C higher. Therefore, the melting temperature of the H-primer can be at least 12 °C higher, e.g., at least 15 °C higher, preferably at least 14 °C higher, even more preferably at least 16 °C higher, even more preferably 18 °C higher, such as at least 20 °C higher, e.g., in the range of 15 to 50 °C, such as in the range of 15 to 40 °C, e.g., in the range of 15 to 25 °C higher than the melting temperature of the L-primer. In some embodiments, it is preferred that the melting temperature of the H-primer is at least 30 °C higher, such as in the range of 30 to 50 °C.

[00131] In general, it is preferred that the H-primer melting temperature be as high as possible, but not above the highest functional elongation temperature of at least one nucleic acid polymerase. Said elongation temperature need not be the optimal temperature for said nucleic acid polymerase, but it is preferred that at least one nucleic acid polymerase have activity at the H-primer melting temperature. Therefore, the H-primer melting temperature may approach or may even exceed 80°C.

[00132] The H-primer may comprise one or more nucleotide analogues, for example any of the nucleotide analogues described herein above in the “Primer Set” section. The incorporation of some nucleotide analogues may increase the melting temperature and accordingly the H-primer may in particular comprise nucleotide analogues, wherein the incorporation of said nucleotide analogues increases the melting temperature of the primer. Therefore, the H-primer may comprise one or more LNAs, PNAs, GNAs and/or TNAs. For example, the H-primer may comprise in the range of 1 to 20, such as in the range of 1 to 15, for example in the range of 5 to 10 nucleotide analogues, for example LNA.

[00133] Since it is also preferred that the L-primer melting temperature be sufficiently high to ensure specific annealing of L-primer to the target nucleic acid sequence/the sequence complementary to the target nucleic acid sequence, and the H-primer melting temperature should be significantly higher than the H-primer melting temperature, then often the H-primer melting temperature is at least 60°C. The H-primer melting temperature may also often be at least 70°C. The H-primer melting temperature may for example be in the range of 60 to 90°C, for example in the range of 60 to 85°C, such as in the range of 70 to 85°C, for example in the range of 70 to 80°C.

[00134] The melting temperature of primer-L is preferably sufficiently high to ensure specific annealing of primer-L to the target nucleic acid sequence/the complementary sequence of the target nucleic acid sequence, but also significantly lower than the melting temperature of primer-H. Often, the melting temperature of primer-L is in the range of 30 to 55 °C, such as in the range of 35 to 55 °C, preferably in the range of 40 to 50 °C.

PCR REAGENTS

[00135] The methods of the invention involve steps of performing PCR. The skilled person is well aware of how to perform a PCR and which reagents may be useful for performing a PCR. Such reagents are referred to as PCR reagents herein. The kit of parts of the invention also comprises PCR reagents.

[00136] For most purposes, PCR reagents comprise nucleotides. Therefore, PCR reagents may comprise deoxynucleoside triphosphates (dNTPs), in particular, all four naturally occurring deoxynucleoside triphosphates (dNTPs).

[00137] PCR reagents often comprise deoxyribonucleoside triphosphate molecules, which include all of dATP, dCTP, dGTP, dTTP. In some cases dUTP is added.

[00138] PCR reagents may also comprise compounds useful in aiding nucleic acid polymerase activity. Thus, the PCR reagent may comprise a divalent cation, for example, magnesium ions. Said magnesium ions may be added in the form of, for example, magnesium chloride or magnesium acetate (MgCl2), or magnesium sulfate is used.

[00139] PCR reagents may also comprise one or more of the following:

[00140] - non-specific blocking agents, such as BSA or bovine hide gelatin, beta-lactoglobulin, casein, milk powder or other common blocking agents,

[00141] - non-specific blocking/background nucleic acids (e.g. salmon sperm DNA),

[00142] - biopreservatives (e.g. sodium azide),

[00143] - PCR enhancers (e.g. Betaine, Trehalose, etc.),

[00144] - inhibitors (e.g. RNAse inhibitors).

[00145] The PCR reagent may also contain other additives, for example, dimethyl sulfoxide (DMSO), glycerol, betaine (mono)hydrate (N,N,N-trimethylglycine=[carboxymethyl]trimethylammonium), trehalose, 7-Deaza-2‘-deoxyguanosine triphosphate (dC7GTP or 7-deaza-2‘-dGTP), formamide (methanamide), tetramethylammonium chloride (TMAC), other tetraalkylammonium derivatives (e.g., tetraethylammonium chloride (TEA-Cl) and tetrapropylammonium chloride (TPrA-Cl), nonionic detergent (e.g., Triton X-100, Tween 20, Nonidet P-40 (NP-40)), or PREXCEL-Q.

[00146] PCR reagents may comprise a buffering agent.

[00147] In some cases, a nonionic Propylene Oxide/Ethylene Oxide block copolymer is added to the aqueous phase at a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. Common biosurfactants include nonionic surfactants such as Pluronic F-68, Tetronics, Zonyl FSN. Pluronic F-68 may be present at a concentration of about 0.5% w/v.

[00148] In some cases, magnesium sulfate can be substituted for magnesium chloride at similar concentrations. A wide range of common commercial PCR buffers from various suppliers can be substituted for the buffered solution.

[00149] The methods of the invention also involve the use of a nucleic acid polymerase and the part kit of the invention also comprises a nucleic acid polymerase. Said nucleic acid polymerase may be any nucleic acid polymerase, such as a DNA polymerase. The nucleic acid polymerase must have activity at the elongation temperature.

[00150] In some embodiments, the nucleic acid polymerase is a DNA polymerase with 5‘ to 3’ exonuclease activity. In particular, this may be the case in embodiments of the invention wherein the methods or kits involve use of a detection probe, such as a Taqman detection probe.

[00151] Any DNA polymerase, for example, a DNA polymerase with 5' to 3' exonuclease activity that catalyzes primer extension may be used. For example, a thermostable DNA polymerase may be used.

[00152] In one embodiment, the nucleic acid polymerase is a Taq polymerase.

PARTITIONING OF PCR REACTIONS

[00153] In general, the methods of the invention comprise a step of preparing partitioned PCR reactions. The partitioned PCR reaction may then be subjected to an AIPR step followed by one or more PCRs as described herein. Preparing partitioned PCR reactions involves dividing the sample into multiple smaller fractions, each comprising a set of primers, nucleic acid polymerase, PCR reagents, and optionally, detection reagents.

[00154] Partitioned PCR reactions can be prepared by a number of different methods. In general, this involves dividing the PCR reactions into physically and spatially separate compartments. Such compartments can be obtained in a variety of ways, for example, the PCR reactions can be partitioned into different vessels. Partitioned PCR reactions can also be prepared by partitioning the PCR reaction into wells of microtiter plates. Partitioned PCR reactions can also be prepared by partitioning the PCR reaction into microwells, microfluidic chambers, capillaries, the dispersed phase of an emulsion, a chamber (e.g., a chamber in a miniature chamber array), a droplet, or a nucleic acid binding surface. Partitioned PCR reactions can also be prepared by partitioning the PCR reaction into discrete spots on a solid support.

[00155] It is preferable that PCR reactions be partitioned in such a way that each partitioned PCR reaction only comprises a small number of template nucleic acids comprising the target nucleic acid sequence. Since the sample is typically randomly distributed in the partitioned PCR reactions, it is possible that some reactions will comprise more template nucleic acids comprising the target nucleic acid sequence than others. Indeed, some of the partitioned PCR reactions may comprise no template nucleic acids comprising the target nucleic acid sequence, while others may comprise multiple copies. If the copies of the template nucleic acid are randomly distributed among the partitions, some partitions should contain no copies, others only one copy, and if the number of partitions is large enough, still others should contain two copies, three copies, and even larger numbers of copies. The probability of finding exactly 0, 1, 2, 3, or more copies in a partition, based on a given average concentration of the template nucleic acid in the partitions, is described by a Poisson distribution. Some samples will not comprise template nucleic acids that comprise the target sequence, and in such an embodiment also none of the partitioned PCR reactions will comprise template nucleic acids that comprise the target sequence.

[00156] In one embodiment, the partitioned PCR reactions each comprise at most 10, such as at most 5, template nucleic acids comprising the target nucleic acid sequence.

[00157] A very useful method for preparing partitioned PCR reactions is to prepare enclosed reaction droplets, in which each droplet contains one partitioned PCR reaction. Therefore, the partitioned PCR reactions can each be contained in droplets prepared using a droplet generator.

[00158] The size of such droplets may vary, but partitioned PCR reactions may, for example, each be contained in a droplet of a volume in the range of 1 to 10,000 picoliters, e.g., approximately 1,000 picoliters.

[00159] The droplets used herein may include emulsion compositions (or mixtures of two or more immiscible fluids) for example as described in U.S. Patent 7,622,280 or as described in the Examples herein below. The droplets may be generated by devices described in WO/2010/036352. The term emulsion as used herein may refer to a mixture of immiscible liquids (such as oil and water). Oil phase and/or water-in-oil emulsions allow compartmentalization of reaction mixtures within the aqueous droplets. The emulsions may comprise aqueous droplets within a continuous oil phase. The emulsions provided herein may be oil-in-water emulsions, wherein the droplets are oil droplets within a continuous aqueous phase. The droplets used in this document are typically designed to prevent mixing between compartments, with each compartment protecting its contents from evaporation and coalescence with the contents of other compartments.

[00160] Droplets may be generated with an average diameter of about, or greater than about, or at least about 0.001; 0.01; 0.05; 0.1; 1; 5; 10; 20; 30; 40; 50; 60; 70; 80; 100; 120; 130; 140; 150; 160; 180; 200; 300; 400, or 500 microns. The droplets may have an average diameter of about 0.001 to about 500, about 0.01 to about 500, about 0.1 to about 500, about 0.1 to about 100, about 0.01 to about 100, or about 1 to about 100 microns. Microfluidic methods of producing emulsion droplets using microchannel cross-flow focusing or physical agitation are known to produce monodisperse or polydisperse emulsions. The droplets may be monodisperse droplets. The droplets may be generated such that the droplet size does not vary by more than or less than 5% of the average droplet size. In some cases, the droplets are generated such that the droplet size does not vary by more than or less than 2% of the average droplet size. A droplet generator can generate a population of droplets from a single sample, in which none of the droplets varies in size by more than or less than about 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of the average size of the total droplet population.

[00161] Superior mechanical stability may be useful for microfluidic manipulations and high-shear fluidic processing (e.g., in microfluidic capillaries or by 90-degree turns, such as valves, in fluidic paths). Pre- or post-heat-treated droplets or capsules may be mechanically stable for standard pipette manipulations and centrifugation.

[00162] Droplets may be polydisperse or monodisperse generated through agitation, sonication or microfluidically through a T-channel junction or other means by those familiar with the art.

[00163] A droplet may be formed by flowing an oil phase through an aqueous sample. The aqueous phase may comprise a buffered solution and reagents for performing a PCR reaction, which include nucleotides, primers, probe(s) for fluorescent detection, template nucleic acids, DNA polymerase enzyme, and optionally reverse transcriptase enzyme.

[00164] The aqueous phase generally comprises the sample, PCR reagents, nucleic acid polymerase, primer set, and optionally detection reagents.

[00165] The oil phase may comprise a fluorinated base oil which may be further stabilized by combination with a fluorinated surfactant such as a perfluorinated polyether. In some cases, the base oil may be one or more of HFE 7500, FC-40, FC-43, FC-70 or another common fluorinated oil. In some cases, the anionic surfactant is Ammonium Krytox (Krytox-AM), the ammonium salt of Krytox FSH, or the morpholino derivative of Krytox-FSH.

[00166] The oil phase may further comprise an additive to adjust oil properties, such as vapor pressure or viscosity or surface tension. Non-limiting examples include perfluorooctanol and 1H,1H,2H,2H-Perfluorodecanol.

[00167] The emulsion may be formulated to produce highly monodisperse droplets having a liquid-like interfacial film that can be converted by heating into microcapsules having a solid-like interfacial film; such microcapsules may behave as bioreactors capable of retaining their contents through a reaction process such as AIPR or PCR amplification. Conversion to the microcapsule form may occur upon heating. For example, such conversion may occur at a temperature of greater than about 50, 60, 70, 80, 90, or 95 degrees Celsius. In some cases, such heating occurs using a thermal cycler. During the heating process, an overlay of fluid or mineral oil may be used to prevent evaporation. Biocompatible capsules may be resistant to coalescence and/or flocculation by a wide range of thermal and mechanical processing.

[00168] In some cases, the droplet is generated using a commercially available droplet generator, such as the Bio-Rad QX100™ Droplet Generator. AIPR and PCR can be performed using commercially available apparatus, and the droplets can be analyzed using a commercially available droplet reader, such as a generator, such as the Bio-Rad QX100™ Droplet Reader.

ASYMMETRICAL INCREMENTAL POLYMERASE REACTION

[00169] The methods of the invention comprise an asymmetric incremental polymerase reaction (AIPR) step. It is important that the AIPR is performed prior to any exponential PCR step. Accordingly, in general, step e) is performed prior to step f). The AIPR comprises the steps of:

[00170] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00171] ii. incubate the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer,

[00172] iii. optionally, incubate the partitioned PCR reactions at the elongation temperature,

[00173] iv. optionally, repeat steps i to iii,

[00174] In general, AIPR is an asymmetric reaction that results in the amplification of only one strand of the target nucleic acid sequence. Therefore, preferably, the PCR reaction does not comprise any other primer, which together with primer-H is capable of amplifying the target nucleic acid sequence, wherein said other primer has a melting temperature similar to or higher than the melting temperature of primer-H. In embodiments of the invention, wherein the primer set comprises multiple primers-H, it is preferable that the PCR reaction does not comprise any other primer, which together with any primer-H is capable of amplifying any target nucleic acid sequences, wherein said other primers have a melting temperature similar to or higher than the melting temperature of primer-H. Preferred melting temperatures of the primers are described in the section “Primer Set” and “Primer-L and Primer-H”. Therefore, at high annealing temperature only primer-H will be annealed resulting in polymerization of only primer-H. Therefore, multiple rounds of AIPR leads to amplification of only one strand of the target nucleic acid sequence, and AIPR is therefore in principle a linear amplification.

[00175] Often, the nucleic acid polymerase has elongation activity at the high annealing temperature. In such embodiments, the high annealing temperature may also be considered to be at the elongation temperature even if the high annealing temperature is not the optimal temperature for the nucleic acid polymerase. Therefore, the AIPR may comprise only two steps that are repeated, i.e., a denaturing step by incubation at the denaturation temperature, and a combined annealing and H-primer elongation step by incubation at the high annealing temperature. In said embodiments, the H-primer is preferably designed to have a melting temperature at a temperature at which the nucleic acid polymerase has sufficient activity to catalyze H-primer elongation.

[00176] Consequently, the AIPR of step e) may comprise the steps of:

[00177] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00178] ii. incubating the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer, wherein the high annealing temperature is also the elongation temperature, which allows extension of the annealed H-primer;

[00179] iii. repeat steps i to ii

[00180] In other embodiments of the invention, the H-initiator melting temperature is different from the elongation temperature. In such embodiments, the AIPR of step e) may comprise the steps of:

[00181] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00182] ii. incubate the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer,

[00183] iii. incubate the partitioned PCR reactions at the elongation temperature, which allows elongation of the annealed H-primer,

[00184] iv. repeat steps i to iii.

[00185] Step i. of incubating the partitioned PCR reactions at a denaturation temperature is done for a sufficiently long time to denature DNA into single-stranded molecules. It is possible that step i. is performed for a longer time during the first cycle of AIPR than in subsequent cycles. The skilled person will be able to select appropriate times for incubation at the denaturation temperature. In the first cycle, incubation at the denaturation temperature may, for example, be in the range of 0.5 to 10 min (e.g. for hot-start DNA polymerases), while in subsequent cycles incubation at the denaturation temperature, for example, may be in the range of 0.1 to 2 min.

[00186] Similarly, the skilled person will be able to select appropriate times for incubation at the high annealing temperature/elongation temperature. In the last cycle, the incubation at the elongation temperature may be longer than other cycles, e.g., in the range of 0.5 to 10 min, while in the other cycles, the incubation at the elongation temperature, for example, may be in the range of 0.1 to 2 min. As outlined above, then the high annealing temperature may be the same as the elongation temperature. In embodiments where the high annealing temperature is different from the elongation temperature, then the incubation at the annealing temperature could, for example, be in the range of 0.1 to 2 min.

[00187] Steps i to ii may be repeated a number of times. In general, steps i to ii are repeated a number of times sufficient to ensure a very low to no occurrence of false positive signals. For example, steps i to ii may be repeated in the range of 8 to 256 times, preferably in the range of 16 to 128 times, for example in the range of 32 to 128 times, for example approximately 64 times, such as 64 times.

[00188] In embodiments of the invention where the high annealing temperature and the elongation temperature are different, then steps i. to iii. may be repeated in the range of 8 to 256 times, preferably in the range of 16 to 128 times, for example in the range of 32 to 128 times, for example approximately 64 times, such as 64 times.

[00189] It is preferred that during AIPR, only incremental copying is performed. In other words, it is preferred that only one strand of the target nucleic acid serves as the template for copying during AIPR. If the H-primer is annealing to a sequence complementary to the target nucleic acid sequence, then it is preferred that only the strand comprising the target nucleic acid sequence is synthesized during AIPR. Since AIPR is monodirectional, said strands may be of different lengths, but they preferably comprise at least the target nucleic acid sequence. In this embodiment, the sequence complementary to the target nucleic acid sequence is preferably not synthesized during the AIPR stage. Similarly, if the H-primer is annealing to the target nucleic acid sequence, then it is preferred that only the strand comprising the sequence complementary to the target nucleic acid sequence is synthesized during AIPR. Since AIPR is monodirectional, said strands may have different lengths, but they preferably comprise at least the sequence complementary to the target nucleic acid sequence. In this embodiment, the target nucleic acid sequence is preferably not amplified during AIPR.

[00190] Therefore, in one embodiment of the invention step e) results in elongation of primer-H but no detectable elongation of primer-L. For example, step e) may result in elongation of primer-H but no elongation of primer-L.

[00191] In one embodiment, step e) results in elongation of primer-H, but no detectable elongation of any other primer. For example, step e) may result in elongation of primer-H, but no elongation of any other primer.

[00192] The high annealing temperature is selected to allow annealing of primer-H but not primer-L. Accordingly, it is preferred that the high annealing temperature is set to be significantly higher than the melting temperature of primer-L. Therefore, the high annealing temperature in step e) may be at least 10°C higher, preferably at least 15°C higher, e.g. at least 20°C, such as at least 25°C higher than the melting temperature of primer-L.

[00193] The high annealing temperature could, for example, be set to approximately the melting temperature of the H-initiator. It could, however, also be somewhat lower.

LOW TEMPERATURE PCR

[00194] In some embodiments of the invention, the methods comprise a low temperature PCR step, which is performed after the completion of AIPR and prior to exponential PCR.

[00195] In such embodiments, the L-primer is typically an alignment-modified L-primer as described herein above.

[00196] This may in particular be the case for difficult targets (e.g. a target with a very homologous sequence elsewhere in the genome, such as a pseudogene), such a misalignment-modified L-primer may allow the use of a shorter L-primer that is more specific to the true target.

[00197] Low temperature PCR typically involves the steps of:

[00198] 1) incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00199] 2) incubate the PCR at a very low annealing temperature that allows annealing of both the H-primer and the unaligned part of the L-primer,

[00200] 3) incubate the PCR at the elongation temperature, which allows extension of all annealed primers

[00201] 4) optionally, repeat steps 1) through 3), which results in a PCR product.

[00202] This step will allow amplification of a product incorporating the misaligned part of primer-L. Once sufficient of said product is available, then a normal exponential PCR can be performed using a low annealing temperature, which for example, may be approximately the melting temperature of primer-L.

[00203] In general, the very low annealing temperature is lower than the low annealing temperature. Typically, the very low annealing temperature is at least 5 °C, preferably at least 10 °C, more preferably at least 15 °C, such as at least 20 °C lower than the low annealing temperature. For example, the very low annealing temperature is in the range of 5 to 30 °C lower than the low annealing temperature, for example, the very low annealing temperature may be in the range of 20 to 25 °C lower than the low annealing temperature. Therefore, the very low annealing temperature may be at least 20 °C lower, for example, at least 25 °C, such as at least 30 °C, for example, at least 35 °C lower than the H-initiator melting temperature.

[00204] Typically, steps 1) to 3) may be repeated more than once, e.g., in the range of 1 to 40. For example, steps 1) to 3) are repeated in the range of 2 to 10 times, e.g., 4 to 6 times. It is often preferred that the total number of PCR cycles performed during low temperature PCR and exponential PCR be in the range of 20 to 40, such as in the range of 20 to 30. Therefore, depending on how many times steps I to III of the exponential PCR are repeated, steps 1) to 3) of the low temperature PCR may be repeated to achieve a total number of cycles in the range of 20 to 40, such as in the range of 20 to 30.

EXPONENTIAL PCR

[00205] The methods of the invention comprise a step of performing a polymerase chain reaction (PCR). In order to discriminate this step from AIPR, this step may also be called “exponential PCR”. In the methods of the invention, the polymerase chain reaction is performed subsequent to AIPR. Exponential PCR may in general comprise the steps of:

[00206] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00207] 2) incubate the PCR at a low annealing temperature that allows both H-primer and L-primer annealing,

[00208] iii. incubate the PCR at the elongation temperature that allows extension of all annealed primers

[00209] iv. optionally, repeat steps I through III, which results in a PCR product.

[00210] Incubation at the low annealing temperature preferentially allows annealing of both primers of the primer pair capable of specifically amplifying the target nucleic acid sequence. Consequently, exponential PCR will result in amplification of both strands of the target nucleic acid sequence. In theory, said amplification will be exponential and can therefore be called “exponential PCR”, although in practice it is possible that it will not be completely exponential.

[00211] Exponential PCR may be performed in any manner, for example, in any conventional manner for performing PCR known to the skilled person.

[00212] Step I. of incubating the partitioned PCR reactions at a denaturation temperature is done long enough to denature DNA into single-stranded molecules. It is possible that step I. is carried out for a longer time during the first cycle of the PCR than in subsequent cycles. The skilled person will be able to select appropriate times for incubation at the denaturation temperature. In the first cycle, incubation at the denaturation temperature may, for example, be in the range of 0.5 to 10 min, while in subsequent cycles incubation at the denaturation temperature, for example, may be in the range of 0.1 to 2 min.

[00213] Similarly, the skilled person will be able to select appropriate times for incubation at the low annealing temperature. For example, incubation at the low annealing temperature could, for example, be in the range of 0.1 to 2 min.

[00214] Similarly, the skilled person will be able to select appropriate times for incubation at the elongation temperature. In the last cycle, the incubation at the elongation temperature may be longer than in the other cycles, for example, in the range of 0.5 to 10 min, while in the other cycles the incubation at the elongation temperature, for example, may be in the range of 0.1 to 2 min.

[00215] Steps I to III may be repeated numerous times. In general, it is preferable that steps I to III are not repeated too many times in order to reduce the risk of false positive signals. For example, step f) may comprise repeating steps I to III in the range of 15 to 60 times, preferably in the range of 20 to 40 times, for example in the range of 20 to 30 times, for example in the range of 25 to 30 times.

[00216] In embodiments of the invention, wherein the primer set comprises additional primers in addition to primer-H and primer-L, then said additional primers in some embodiments may also anneal to their template at the low annealing temperature.

[00217] Therefore, in one embodiment of the invention, step f) results in the elongation of primer-H and primer-L. In another embodiment of the invention, step f) results in the elongation of all primers in the primer set.

[00218] The low annealing temperature is selected so that primer-L can anneale to the target nucleic acid sequence. Therefore, the low annealing temperature could, for example, be set to be approximately the melting temperature of primer-L. It could, however, also be somewhat lower.

[00219] Examples of PCR techniques that can be used for exponential PCR include, but are not limited to, quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplexing fluorescent PCR (MF-PCR), real-time PCR (RT-PCR), single-cell PCR, restriction fragment length polymorphism PCR (PCR-RFLP), PCR-RFLP/RT-PCR-RFLP, hot start PCR, nested PCR, polymerase in situ colony PCR, rolling circle amplification (RCA), digital PCR (dPCR), droplet digital PCR (ddPCR), bridging PCR, picotiter PCR, and emulsion PCR.

DETECTION

[00220] In general, the methods of the invention comprise a step of detecting whether the PCR product comprises the variant sequence and/or the target nucleic acid sequence. Said detection may be achieved in any suitable manner known to the skilled person. For example, numerous useful detection methods are known in the prior art that may be employed with the methods of the invention.

[00221] In one embodiment of the invention, said detection involves the partitioned PCR reactions containing a detection reagent. Said detection reagent may be any detectable reagent, for example, it may be a compound comprising a detectable identifier, wherein said detectable identifier, for example, may be a dye, a radioactive activity, a fluorophore, a heavy metal, or any other detectable identifier.

[00222] Often, the detection reagent comprises a fluorescent compound.

[00223] In one embodiment, the detection reagent of the invention comprises or consists of detection probes. The detection probes preferably comprise or consist of nucleotide oligomers or polymers which optionally may comprise nucleotide analogues, such as any of the nucleotide analogues described herein above in the “Primer Set” section. Often, the detection probe may be a DNA oligomer. Typically, the detection probe is linked to a detectable label, e.g., by a covalent bond. The detectable label may be any of the labels mentioned above, but is often a fluorophore. It is preferred that the probe not be capable specifically of amplifying a target nucleic acid together with Primer-H. For example, if Primer-H comprises a sequence identical to a fragment of the target sequence, then the probe(s) may comprise a sequence identical to another fragment of the target sequence. If the H-primer comprises a sequence complementary to one fragment of the target sequence, then the probe(s) may comprise a sequence complementary to another fragment of the target sequence.

[00224] In general, the detection probe is capable of specifically binding to the target nucleic acid sequence. For example, the detection probe may be capable of specifically binding to the target nucleic acid comprising the variant sequence. Therefore, the detection probe may be capable of annealing to the target nucleic acid sequence or to the sequence complementary to the target nucleic acid sequence. Therefore, the detection probe may comprise a sequence identical to a fragment of the target nucleic acid sequence or to the sequence complementary to the target nucleic acid sequence. It is generally preferred that the detection probe comprise a sequence different from the sequence of any of the primers in the primer set.

[00225] The detection probe(s) is designed to have an appropriate melting temperature. In one embodiment, the melting temperature of at least one detection probe is significantly lower than the melting temperature of H-initiator. For example, the melting temperature of all detection probes is significantly lower than the melting temperature of H-initiator. Therefore, the melting temperature of at least one detection probe may be at least 12°C lower, preferably at least 14°C lower, even more preferably at least 16°C lower, even more preferably 18°C lower, such as at least 20°C lower, for example in the range of 15 to 25°C, such as in the range of 30 to 40°C, or even up to 45°C lower than the melting temperature of H-initiator. For example, the melting temperature of all detection probes may be at least 12 °C lower, preferably at least 14 °C lower, even more preferably at least 16 °C lower, even more preferably 18 °C lower, such as at least 20 °C lower, for example in the range of 15 to 45 °C lower than the melting temperature of the H-initiator. However, probes with a higher melting temperature may also be applied.

[00226] The melting temperature of the detection probe(s) is preferably high enough to ensure specific annealing of detection probes, but also significantly lower than the melting temperature of Primer-H. Often, the melting temperature of the detection probes can be similar to the melting temperature of Primer-L. For example, the melting temperature of the at least one detection probe can be the same as the melting temperature of Primer-L +/- 10 °C, such as the same as the melting temperature of Primer-L +/- 5 °C, e.g., approximately the same as the melting temperature of Primer-L. In other embodiments, the melting temperature of the probe can be higher than the melting temperature of Primer-L, e.g., 15 to 20 °C higher than the melting temperature of Primer-L. Therefore, the detection probe may, for example, have a melting temperature in the range of 35 to 60 °C, such as in the range of 35 to 55 °C, preferably in the range of 40 to 50 °C.

[00227] It is often preferable for the detection probe to provide a different detectable signal depending on the presence of the target nucleic acid sequence. This can be achieved in a number of different ways.

[00228] In one embodiment, the detection probe is linked to at least one fluorophore and at least one quencher capable of quenching signal from the fluorophore when said detection probe is not bound to its target. Consequently, fluorescence from said fluorophore will not be detectable. However, if the detection probe binds to the target nucleic acid sequence/the sequence complementary to the target nucleic acid sequence, then this causes the quencher to be sufficiently removed from the fluorophore such that the abolished quenching allows fluorescence from the fluorophore to be detected. Removing the quencher from the fluorophore can be achieved in a number of ways. For example, PCR can employ the use of a nucleic acid polymerase that has 5' to 3' exonuclease activity. Upon elongation, any bound probe will be degraded by said 5' to 3' exonuclease activity that separates the fluorophore from the quencher. It is also possible that the detection probe changes 3D conformation upon binding to the quencher that is removed from the fluorophore.

[00229] In one embodiment, the partitioned PCR reactions of the invention each contain a detection reagent, which is a variant detection probe. The variant detection probe is a detection probe, as described above, that is capable of hybridizing to the target nucleic acid sequence containing the variant sequence with significantly greater affinity than to the target nucleic acid sequence not containing the variant sequence.

[00230] In one embodiment of the invention, the partitioned PCR reactions each contain a detection reagent that is a wild-type detection probe. The wild-type detection probe is a detection probe as described above that is capable of hybridizing to the target nucleic acid sequence that does not contain the variant sequence. It may be preferred that the wild-type detection probe be capable of hybridizing to the target nucleic acid sequence that does not contain the variant sequence with significantly greater affinity than to the target nucleic acid sequence that contains the variant sequence. In embodiments related to detecting the presence of a target nucleic acid sequence, then the partitioned PCR reactions may each contain a detection reagent that is a wild-type detection probe. In such embodiments, it may be sufficient for the partitioned PCR reactions to contain only one type of detection probe. For example, PCR reactions may contain a wild-type detection probe as the sole detection probe or PCR reactions may contain variant detection probe as the sole detection probe.

[00231] In one embodiment of the invention, each partitioned PCR reaction contains both a variant detection probe and a wild-type detection probe. In particular, this may be the case in embodiments relating to the detection of a variant sequence in a target nucleic acid sequence.

[00232] The variant detection probe may be linked to at least one fluorophore and at least one quencher, wherein the quencher is capable of quenching the fluorescence of the fluorophore. Preferably, the quencher and the fluorophore are linked to the variant detection probe in a manner in which the quencher is capable of quenching the fluorescence of the fluorophore when the probe is present in its free state. Therefore, often, the fluorophore and the quencher are positioned close to each other such that the quencher is capable of quenching the fluorescence of the fluorophore. Often, the fluorophore and the quencher are linked to different nucleotides in the variant detection probe.

[00233] The wild-type detection probe may be linked to at least one fluorophore and at least one quencher, the quencher being capable of quenching the fluorescence of the fluorophore. Preferably, the quencher and the fluorophore are linked to the wild-type detection probe in a manner in which the quencher is capable of quenching the fluorescence of the fluorophore when the probe is present in its free state. Therefore, often, the fluorophore and the quencher are positioned close to each other such that the quencher is capable of quenching the fluorescence of the fluorophore. Often, the fluorophore and the quencher are linked to different nucleotides in the wild-type detection probe.

[00234] In embodiments of the invention, both a variant detection probe and a wild-type detection probe are employed, whereby the variant detection probe may be linked to a different fluorophore than the wild-type detection probe. In particular, the variant detection probe may be linked to at least one fluorophore that has fluorescence that is distinguishable from the fluorescence of all fluorophores linked to the wild-type detection probe. Similarly, the variant wild-type detection probe may be linked to at least one fluorophore that has fluorescence that is distinguishable from the fluorescence of all fluorophores linked to the variant detection probe.

[00235] Step g) of the methods of the invention may involve detecting fluorescence from the variant detection probe. Therefore, step g) may comprise detecting fluorescence from the fluorophore bound to the variant detection probe. This may be the case in embodiments of the invention, for example, wherein:

[00236] • the variant probe is linked to at least one fluorophore, and at least one quencher, capable of quenching the fluorescence of the fluorophore;

[00237] • nucleic acid polymerase is a DNA polymerase that has 5‘ to 3‘ exonuclease activity; and/or

[00238] • the method is the method of detecting the presence of a variant sequence in a target nucleic acid sequence.

[00239] Similarly, step g) of the methods of the invention may involve detecting fluorescence from the wild-type detection probe. Therefore, step g) may comprise detecting fluorescence from the fluorophore bound to the wild-type detection probe. This may be the case in embodiments of the invention, for example, wherein:

[00240] • the wild-type detection probe is linked to at least one fluorophore, and at least one quencher, capable of quenching the fluorescence of the fluorophore;

[00241] • nucleic acid polymerase is a DNA polymerase that has 5‘ to 3‘ exonuclease activity; and/or

[00242] • the method is a method of detecting the presence of the target nucleic acid sequence.

[00243] In embodiments of the invention, where the PCR is partitioned into PCR reactions contained in droplets, then said fluorescence may, for example, be detected using a detector that has handling capabilities for droplet samples, with individual droplets entering the detector and subjected to detection, then exiting the detector. For example, a flow cytometry device may be adapted for use in detecting fluorescence from droplet samples. In some cases, a microfluidic device equipped with pumps to control droplet movement is used to detect fluorescence from droplets in a single file. In some cases, droplets are arranged on a two-dimensional surface and a detector moves relative to the surface, detecting fluorescence at each position containing a single droplet.

[00244] Following acquisition of fluorescence detection data, a computer may be used to store and process the data. Computer executable logic may be employed to perform such functions as subtraction of background fluorescence, assignment of target and/or reference sequences, and quantification of the data. A computer may be useful for displaying, storing, retrieving, or calculating diagnostic results from molecular profiling; displaying, storing, retrieving, or calculating raw data from genomic or acid expression analysis; or displaying, storing, retrieving, or calculating any sample or patient information in the methods described herein.

[00245] The detection signal(s) may be generated based on detected light emitted by the wild-type detection probe and, optionally, the variant detection probe in the partitions. The variant detection probes may report whether at least one of two or more particular amplification reactions represented by the signal has occurred in a partition and, therefore, whether at least one copy of the variant sequence is present in the partition. The level or amplitude of the signal corresponding to the probes may be analyzed to determine whether or not at least one of the particular reactions has occurred and at least one copy of one of the particular targets is present. The level or amplitude of the signal may vary between partitions according to the presence or absence of the target nucleic acid sequence in each partition. For example, a partition positive for a particular target may produce a signal level or amplitude that is above a given threshold and/or within a given range. Partitions may be analyzed and signals created at any suitable time(s). Exemplary time points include at the end of an assay (assay endpoint), when reactions have been completed and the data are no longer changing, or at an earlier time point, provided that the data are sufficiently and reliably separated. In general, it may be preferable to perform detection after performing the number of exponential PCR cycles necessary for true positive signals to be of sufficiently high amplitude.

[00246] In one aspect, a method for detecting the presence of a target sequence using a single detection probe is provided herein.

[00247] In some cases, AIPR and exponential PCR are performed in a dPCR setup, such as ddPCR. Two distinct droplet populations may be detectable and counted to determine the concentration of target nucleic acid sequence comprising a variant sequence (see overview in Figure 1A) versus that not comprising said variant sequence (see Figure 1B).

[00248] Fluorophore as used herein may mean a compound with fluorescent emission, for example, with a maximum fluorescent emission between about 350 and about 900 nm. A wide variety of fluorophores may be used, which include but are not limited to: 5-FAM (also called 5-carboxyfluorescein; also called Spiro(isobenzofuran-1(3H),9′-(9H)xanthene)-5-carboxylic acid,3′,6′-dihydroxy-3-oxo-6-carboxyfluorescein); 5-Hexachloro-Fluorescein; ([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6-Hexachloro-Fluorescein; ([4,7,2‘,4‘,5‘,7‘-hexachloro-(3‘,6‘-dipivaloylfluoresceinyl)-5-carboxylic]); 5-Tetrachloro-Fluorescein; ([4,7,2‘,7‘-tetrachloro-(3‘,6‘-dipivaloylfluoresceinyl)-5-carboxylic] acid); 6-Tetrachloro-Fluorescein; ([4,7,2‘,7‘-tetrachloro-(3‘,6‘-dipivaloylfluoresceinyl)-6-carboxylic] acid); 5-TAMRA(5-carboxytetramethylrhodamine); Xanthilium, 9-(2,4-dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA (6-carboxytetramethylrhodamine); 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid); 1,5-IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid); Cy5 (Indodicarbocyanine-5); Cy3 (Indodicarbocyanine-3); and BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid); Quasar™-670 dye (Biosearch Technologies); Cal Fluor™ Orange dye (Biosearch Technologies); Rox dyes; Max dyes (Integrated DNA Technologies) as well as suitable derivatives thereof.

[00249] Quencher, as used herein, may mean a molecule or part of a compound that has the ability to reduce the fluorescence of a fluorophore when attached to or in proximity to the detection probe. Quenching may occur through any of a number of mechanisms including fluorescence resonance energy transfer, photoinduced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange coupling, and exciton coupling such as the formation of dark complexes. The selection of the quencher may depend on the fluorophore used. Numerous commercially available quenchers are known in the art and include, but are not limited to, DABCYL, Black Hole™ (BHQ-1, BHQ-2, and BHQ-3), Iowa Black™ FQ, and Iowa Black™ RQ quenchers. These are referred to as dark quenchers. They do not fluoresce, which can eliminate the background seen with other quenchers such as TAMRA that is intrinsically fluorescent.

[00250] There is a good deal of practical guidance available in the literature for selecting appropriate quencher-reporter pairs for particular probes, as exemplified by the following references: Clegg, Meth. Enzymol., 211:353-388 (1992); Wo et al., Anal. Biochem., 218:1-13 (1994); Pesce et al., eds., Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); and the like. The literature also includes references providing exhaustive lists of fluorescent and chromogenic molecules and their optical properties relevant to choosing quencher-reporter pairs, e.g., Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color and Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); and the like. Additionally, there is extensive guidance in the literature for deriving reporter and quencher molecules for covalent attachment via common reactive groups that can be added to an oligonucleotide, as exemplified by the following references: Haugland (cited above); Ullman et al., U.S. Patent 3,996,345; Khanna et al., U.S. Patent 4,351,760.

[00251] Both the detectable label (e.g., the fluorophore) and the quencher can be attached to the probe using methods known in the art. In some cases, one of the reporter/quencher pair is attached to the 5′ portion of a probe and 5′ to the target locus if the probe sequence is complementary to the target locus, and the other of the reporter/quencher pair is attached to the 3′ portion of the probe.

[00252] Detectable labels and quenchers can be added during oligonucleotide synthesis via standard phosphoramidite chemistry. They can also be added post-synthesis by introducing a linker with an appropriate functional group during oligosynthesis. Following synthesis, a detectable (e.g., fluorophore) can be coupled to an oligonucleotide functional group. For longer sequences, to allow for efficient quenching, the sequence immediately 3' of the fluorophore and 5' of the quencher can be made complementary to each other to allow for the formation of a hairpin stem (e.g., molecular beacon). Therefore, during the annealing phase of AIPR and/or exponential PCR, such a probe will hybridize to the amplified target sequence, which physically distances the detectable label (e.g., fluorophore) from the quencher allowing greater fluorescence to be detected. However, in the absence of amplified target sequence, the probe creates a clamp that brings the detectable (e.g., fluorophore) and quencher into close proximity to each other, which limits the fluorescence of the reaction. In these reactions, a polymerase with 5'-3' exonuclease activity is not required to cleave the probe. The appropriate attachment site for the reporter signal (e.g., fluorophore) and quencher and the distance between the reporter signal (e.g., fluorophore) and quencher are known in the art.

[00253] In some cases, the detection probe is a TaqMan® probe.

[00254] A TaqMan® probe (Heid et. al, 1996) can use the fluorogenic 5′ exonuclease activity of Taq polymerase to measure the amount of target sequences in cDNA samples. TaqMan® probes may contain a fluorophore usually at or near the 5′ base, and a quencher may be at or near the 3′ base. The quencher may be a dye such as TAMRA or may be a nonfluorescent molecule such as 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL). See Tyagi et al., Nature Biotechnology 16:49–53 (1998). When irradiated, the excited fluorescent dye transfers energy to the nearby quenching dye molecule instead of fluorescing (termed FRET=fluorescence or Forster resonance energy transfer). Therefore, the proximity of the fluorophore and quencher can prevent any fluorescence from being emitted while the probe is intact. TaqMan® probes can be designed to anneal to an internal region of the PCR product. When the polymerase replicates a template to which a TaqMan® probe is attached, its 5′ exonuclease activity can cleave the probe. This cleavage can terminate the quencher activity (no FRET) and the reporter dye begins to emit fluorescence that can increase with each cycle proportional to the rate of probe cleavage. Accumulation of PCR product can be detected by monitoring the increase in fluorescence of the fluorophore. Because cleavage can occur if the detection probe hybridizes to the target nucleic acid sequence, the detected fluorescence may originate from specific amplification. In some cases, the hybridization and cleavage process do not interfere with exponential PCR. In some cases, a fluorogenic probe does not have G at the 5' end. A and G adjacent to the reporter dye can quench reporter fluorescence even after cleavage.

[00255] In some cases, the detection probe is a molecular beacon. Molecular beacons (MBs) may be oligonucleotides designed for the detection and quantification of target nucleic acids (e.g., target DNAs). The 5' and 3' termini of an MB may collectively comprise a pair of chemical moieties that can confer detectable properties on the MB. One terminus may be attached to a fluorophore and the other may be attached to a quencher molecule capable of quenching fluorescent emission from the fluorophore. For example, a fluorophore/quencher pair may use a fluorophore such as EDANS or fluorescein, for example, at the 5' end and a quencher such as Dabcyl, for example, at the 3' end.

[00256] When an MB is present free in solution, i.e., not hybridized to a second nucleic acid, the stem of the MB can be stabilized by complementary base pairing. This self-complementary base pairing can result in a “hairpin loop” structure for the MB in which the fluorophore and quencher chemical moieties are in close proximity to each other. In this confirmation, the fluorescent chemical moiety can be quenched by the fluorophore.

[00257] The molecular beacon loop may be complementary or identical to part of the target nucleic acid sequence, such that hybridization of the loop to its complementary sequence on the target forces dissociation from the stem, which distances the fluorophore and quencher from each other. This distancing may result in de-quenching of the fluorophore, which causes an increase in MB fluorescence.

[00258] Additional details regarding standard methods of manufacturing and using MBs are well established in the literature, for example in Leone et al. (1995) “Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA.” Nucleic Acids Res. 26:2150–2155; Tyagi and Kramer (1996) “Molecular beacons: probes that fluoresce upon hybridization” Nature Biotechnology 14:303–308; Blok and Kramer (1997), U.S. Patent 6,548,254.

[00259] The detection probe may also be a Scorpions™ probe. A Scorpions™ probe may provide a FRET-based stem-loop detection mechanism similar to Molecular Beacon, except that the probe also has a fixed segment that serves as an amplification primer (see e.g., Whitcombe et al. Nat. Biotechnol. 1999, August 17(8): 804-807; U.S. Patent 6,326,145). In some cases, the probe may be a Sunrise™ probe. A Sunrise™ probe may comprise a primer attached to a hairpin probe that is extended during amplification. This arrangement may separate the identification of the internal quencher from the 5′-terminal fluorophore (Nazarenko et al., Nucl. Acids Res. 1997, 25: 2516-2521).

[00260] The 3' terminal nucleotide of the oligonucleotide probe may be blocked or rendered incapable of extension by a nucleic acid polymerase. Such blocking may be conveniently accomplished by attaching a reporter or quencher molecule to the 3' terminal carbon of the oligonucleotide probe by a chemical linker moiety.

[00261] In some cases, a reference probe may be included in the partitioned PCR reactions. A reference probe may be a non-specific reference probe or a specific reference probe. The reference probe may hybridize to a reference locus.

[00262] In one embodiment, the methods of the invention involve using a single primer pair consisting of primer-H and primer-L combined with a detection probe, such as a variant detection probe or a wild-type detection probe. In other embodiments of the invention, the methods involve using a single primer pair consisting of primer-H and primer-L combined with two detection probes that are a variant detection probe and a wild-type detection probe.

[00263] In one embodiment, detection is performed with the aid of a detection reagent that is a dye. For example, the dye may be a major groove binder, a minor groove binder, an intercalator, or an external binder, among others. Exemplary dyes that may be suitable include luminescent cyanines, phenanthridines, acridines, indoles, imidazoles, and the like, such as DAPI, Hoechst® 33258 dye, acridine orange, etc. Exemplary intercalated dyes that may be suitable include ethidium bromide, propidium iodide, EvaGreen® dye, SYBR® green dye, SYBR® Gold dye, and 7-aminoactinomycin D (7-AAD) among others.

[00264] As used herein, “a probe capable of detecting a specific mutation” is typically a probe comprising a consecutive sequence of the target sequence comprising said mutation or a sequence complementary thereto.

METHOD FOR PREDICTING THE PRESENCE OF A CLINICAL CONDITION

[00265] The methods of the invention may have a multitude of applications. Indeed, the methods are useful in any application in which it is desirable to detect the presence of a target nucleic acid sequence, and/or to distinguish between target nucleic acid sequences that do or do not comprise a variant sequence.

[00266] For example, the methods may be useful for forensic applications, where analysis of nucleic acids in very limited material is often performed. For example, the methods may be used in preparing profiles of genetic material to determine the presence of particular polymorphisms.

[00267] A very useful application of the methods of the invention is to predict the presence of a clinical condition in an individual.

[00268] Many clinical conditions are associated with the presence of particular target nucleic acid sequences. Some clinical conditions are characterized by the presence of a variant sequence in a target nucleic acid. Other clinical conditions are associated with markers, e.g., the presence of a variant sequence may be an indicator of clinical conditions.

[00269] Therefore, in one embodiment, the invention relates to methods of predicting the presence of a clinical condition in an individual, wherein said clinical condition is linked to the presence of a variant sequence in a target nucleic acid sequence, said method comprising the steps of

[00270] a) providing a sample from said individual comprising model nucleic acids

[00271] b) performing the methods of detecting a variant sequence in a target nucleic acid sequence described in this document

[00272] wherein the presence of said variant sequence in said target nucleic acid is indicative of the presence of said clinical condition.

[00273] Many clinical conditions are associated with the presence of one or more variant sequences. Therefore, a clinical condition may be any clinical condition linked to the presence of a variant sequence in a target nucleic acid sequence.

[00274] The variant sequence may be a biomarker that correlates, for example, with the presence of a clinical condition, the risk of progression of a clinical condition, the susceptibility of the clinical condition to a particular treatment, or the risk of death. Therefore, the variant sequence may correlate with a prediction regarding a clinical condition.

[00275] In one embodiment of the invention, the clinical condition is cancer. Said cancer may be any cancer, for example, a cancer selected from the group consisting of carcinoma of the breast, colorectal, pancreas, stomach, GIST, hepatocellular, lung, small cell lung, ovarian, uterine, cervix, bladder, renal, prostate, testis, thyroid carcinoma, malignant melanoma, osteosarcoma, chondrosarcoma, myosarcoma, glioblastoma or other brain tumors, head/neck other gastrointestinal and germ cell tumors, and hematologic malignancies.

[00276] The variant sequence may be linked to said cancer. For example, the presence of the variant sequence may be indicative of the presence of said cancer. However, often additional investigation will be necessary to determine whether said individual is developing said cancer.

[00277] The table below provides non-limiting examples of cancer-linked mutations whose presence may be detected using the methods of the invention. However, the skilled person will be aware of numerous other cancer-linked mutations that may be detected using the methods of the invention.

[00278] The above mutations are indicated at the protein level. The first letter indicates the wild-type amino acid, the number is the position of the amino acid and the last letter the replacement amino acid found in the mutant. Thus, as an example H1047R indicates that histidine at amino acid number 1047 has been replaced by arginine. IUPAC one-letter or three-letter codes for amino acids are used in this document.

[00279] The invention may therefore relate to methods and part kits comprising (using) a pair of primers, wherein

[00280] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of SEQ ID NO:69, 70, 71, or 72 and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of the complementary sequence to SEQ ID NO:69, 70, 71, or 72; OR

[00281] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of the complementary sequence to SEQ ID NO:69, 70, 71 or 72 and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of SEQ ID NO:69, 70, 71 or 72;

[00282] and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising at least one of the nucleotides encoding any of the mutations mentioned in this document.

[00283] In one embodiment, the invention provides methods of predicting the presence of a clinical condition in an individual, wherein said clinical condition is linked to the presence of a target nucleic acid sequence, said method comprising the steps of

[00284] a) providing a sample from said individual comprising model nucleic acids

[00285] b) performing the methods of detecting a variant sequence in a target nucleic acid sequence described in this document

[00286] wherein the presence of said target nucleic acid is indicative of the presence of said clinical condition.

[00287] The clinical condition is infection with an infectious pathogen in which case the target nucleic acid, for example, may be a nucleic acid sequence from the genome of said pathogen.

[00288] The sample may be any sample from said individual. In particular, the sample should be a sample comprising template nucleic acids. In embodiments of the invention where the clinical condition is cancer, it is preferred that the sample comprises DNA from cancer cells. Therefore, the sample may comprise cancer cells comprising DNA and/or the sample may comprise free DNA derived from cancer cells.

[00289] The sample may, for example, be selected from the group consisting of blood samples, biopsy samples, stool samples, saliva samples, urine samples, vaginal fluid samples, ascites fluid samples, cerebrospinal fluid samples, and tissue exudate samples. The sample may also be a fraction of any of the above. For example, the sample may be a blood sample or a fraction thereof, such as a plasma sample or a serum sample.

[00290] The template nucleic acids may be any nucleic acid comprised in the sample, for example, genomic DNA or RNA. The template nucleic acids may also be cDNA prepared based on RNA present in the sample.

[00291] In one embodiment, the template nucleic acids are selected from the group consisting of cell-free DNA, nucleosome DNA, and circulating tumor DNA (ctDNA) that can be found in the blood circulation and other bodily fluids.

[00292] The sample according to the present invention can be extracted from an individual and used for the methods of the invention.

[00293] The individual may be any animal, such as a mammal, which includes humans. In a preferred embodiment, the individual is a human.

EXAMPLES

[00294] The invention is further illustrated by the following examples which, however, should not be construed as limiting the invention.

ITEMS

[00295] The invention may be further defined by the following items:

[00296] 1. A method for detecting the presence of a variant sequence in a target nucleic acid sequence in a sample comprising the steps of

[00297] a) providing a sample comprising model nucleic acids

[00298] b) providing a primer set comprising at least one pair of primers capable specifically of amplifying the target nucleic acid sequence, wherein the primer set comprises at least one H-primer and one L-primer, wherein the melting temperature of H-primer is at least 10 °C higher than the melting temperature of L-primer, and wherein the L-primer contains a sequence complementary to a fragment of the elongation product of H-primer,

[00299] c) providing a nucleic acid polymerase that has polymerase activity at an elongation temperature,

[00300] d) preparing partitioned PCR reactions, each comprising a portion of the sample, the primer set, the nucleic acid polymerase, PCR reagents and, optionally, detection reagents

[00301] e) perform an asymmetric incremental polymerase reaction (AIPR) comprising the steps of:

[00302] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00303] ii. incubate the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer,

[00304] iii. optionally, incubate the partitioned PCR reactions at the elongation temperature,

[00305] iv. optionally, repeat steps i to iii,

[00306] f) perform a polymerase chain reaction (PCR) comprising the steps of:

[00307] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00308] ii. incubate the PCR at a low annealing temperature that allows both H-primer and L-primer annealing,

[00309] iii. incubate the PCR at the elongation temperature that allows extension of all annealed primers

[00310] iv. optionally, repeat steps II through IV, which results in a PCR product.

[00311] g) detecting whether the PCR product comprises the variant sequence in the target nucleic acid sequence.

[00312] 2. The method according to item 1, wherein step e) results in the amplification of only one strand of the target nucleic acid sequence.

[00313] 3. The method according to any of the preceding items, wherein step f) results in the amplification of both strands of the target nucleic acid sequence, which results in a PCR product.

[00314] 4. The method of any of the preceding items, wherein the variant sequence is a single nucleotide mutation.

[00315] 5. The method of any one of items 1 to 3, wherein the variant sequence is a single nucleotide polymorphism (SNP).

[00316] 6. The method of any of the preceding items, wherein the PCR product may comprise both the target nucleic acid sequence comprising the variant sequence and the target nucleic acid sequence lacking the variant sequence.

[00317] 7. A method for detecting the presence of a target nucleic acid sequence in a sample comprising the steps of

[00318] a) providing a sample comprising model nucleic acids

[00319] b) providing a primer set comprising at least one pair of primers capable specifically of amplifying the target nucleic acid sequence, wherein the primer set comprises at least one H-primer and one L-primer, wherein the melting temperature of H-primer is at least 10 °C higher than the melting temperature of L-primer, and wherein the L-primer contains a sequence complementary to the elongation product of H-primer,

[00320] c) providing a nucleic acid polymerase that has polymerase activity at an elongation temperature that is higher than the H-primer melting temperature,

[00321] d) preparing partitioned PCR reactions, each comprising a portion of the sample, the primer set, the nucleic acid polymerase, PCR reagents and, optionally, detection reagents

[00322] e) perform an asymmetric incremental polymerase reaction (AIPR) comprising the steps of:

[00323] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00324] ii. incubate the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer,

[00325] iii. optionally, incubate the partitioned PCR reactions at the elongation temperature,

[00326] iv. optionally, repeat steps i to iii,

[00327] f) perform a polymerase chain reaction (PCR) comprising the steps of:

[00328] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00329] ii. incubate the PCR at a low annealing temperature that allows both H-primer and L-primer annealing,

[00330] iii. incubate the PCR at the elongation temperature that allows extension of all annealed primers

[00331] iv. optionally, repeat steps II through IV, which results in a PCR product.

[00332] g) detecting whether the PCR product comprises the target nucleic acid sequence.

[00333] 8. The method according to any of the preceding items, wherein the AIPR of step e) comprises the steps of:

[00334] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00335] ii. incubating the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer, wherein the high annealing temperature is also the elongation temperature, which allows extension of the annealed H-primer;

[00336] iii. repeat steps i to ii

[00337] 9. The method according to any one of items 1 to 7, wherein the AIPR of step e) comprises the steps of:

[00338] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00339] ii. incubate the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer,

[00340] iii. incubate the partitioned PCR reactions at the elongation temperature, which allows elongation of the annealed H-primer,

[00341] iv. repeat steps i to iii.

[00342] 10. The method of any of the preceding items, wherein primer-H is the only primer in the primer set that has a melting temperature at least 10 °C higher than the melting temperature of primer-L.

[00343] 11. The method according to any of the preceding items, wherein the primer set consists of the H-primer and the L-primer, and wherein the H-primer and the L-primer are capable specifically of amplifying the target nucleic acid sequence.

[00344] 12. The method of any of the preceding items, wherein primer-H is identical to the sequence at the 5' end of the target nucleic acid sequence and primer-L is identical to the complementary sequence at the 3' end of the target nucleic acid sequence.

[00345] 13. The method of any of the preceding items, wherein step e) results in elongation of primer-H but no detectable elongation of primer-L.

[00346] 14. The method of any of the preceding items, wherein step e) results in elongation of primer-H but no detectable elongation of any other primer.

[00347] 15. The method of any of the preceding items, wherein the melting temperature of initiator-H is at least 15 °C higher than the melting temperature of initiator-L.

[00348] 16. The method according to any of the preceding items, wherein the melting temperature of the H-initiator is at least 16 °C higher, preferably at least 18 °C higher, such as at least 20 °C higher, for example in the range of 15 to 50 °C, such as in the range of 15 to 25 °C higher than the melting temperature of the L-initiator.

[00349] 17. The method according to any of the preceding items, wherein the melting temperature of the initiator-H is in the range of 60 to 90 °C, for example in the range of 60 to 80 °C, preferably in the range of 70 to 85 °C, such as in the range of 70 to 80 °C.

[00350] 18. The method according to any of the preceding items, wherein the melting temperature of the initiator-L is in the range of 30 to 55 °C, such as in the range of 35 to 55 °C, preferably in the range of 40 to 50 °C.

[00351] 19. The method according to any of the preceding items, wherein Primer-H has a melting temperature at least 15 °C higher than the melting temperature of any other primer within the primer set that together with Primer-H is capable of amplifying the target nucleic acid sequence

[00352] 20. The method of any of the preceding items, wherein the primer set does not comprise any primers:

[00353] a) having a melting temperature that is in the range of +/- 15 °C, preferably in the range of +/- 20 °C, such as in the range of +/- 25 °C of the H-initiator melting temperature; and

[00354] b) which, together with the H-primer, can constitute a pair of primers capable specifically of amplifying the target nucleic acid sequence.

[00355] 21. The method of any of the preceding items, wherein the primer set comprises more than one H-primer, and any primer that together with any of the H-primer forms a primer pair has a melting temperature that is at least 15 °C lower than the melting temperature of the H-primer of that primer pair.

[00356] 22. The method of any of the preceding items, wherein the H-primer comprises one or more nucleotide analogues, for example, one or more LNAs.

[00357] 23. The method according to any of the preceding items, wherein the partitioned PCR reactions each comprise on average at most 10, such as at most 5, template nucleic acids comprising the target nucleic acid sequence.

[00358] 24. The method of any of the preceding items, wherein the partitioned PCR reactions are each contained in droplets prepared using a droplet generator.

[00359] 25. The method of any of the preceding items, wherein the partitioned PCR reactions are each contained in a volume in the range of 1 to 10,000 picoliters, e.g., approximately 1,000 picoliters.

[00360] 26. The method of any one of items 1 to 23, wherein the partitioned PCR reactions are contained in microtiter plates.

[00361] 27. The method according to any of the preceding items, wherein step e) comprises repeating steps i. to iii. in the range of 8 to 256 times, preferably in the range of 16 to 128 times, for example in the range of 32 to 128 times, for example approximately 64 times, such as 64 times.

[00362] 28. The method according to any one of items 9 and 10 to 27, wherein step e) comprises repeating steps i. to iii. in the range of 8 to 256 times, preferably in the range of 16 to 128 times, for example in the range of 32 to 128 times, for example approximately 64 times, such as 64 times.

[00363] 29. The method according to any of the preceding items, wherein the high annealing temperature in step e) is at least 10 °C higher, preferably at least 15 °C higher, for example at least 20 °C higher, than the melting temperature of initiator-L.

[00364] 30. The method according to any of the preceding items, wherein step f) comprises repeating steps I. to III. in the range of 15 to 60 times, preferably in the range of 20 to 40 times, for example in the range of 20 to 30 times, for example in the range of 25 to 30 times.

[00365] 31. The method according to any of the preceding items, wherein the L-primer is a misaligned modified L-primer and the method comprises a low temperature PCR step between steps e) and f), wherein the low temperature PCR comprises the steps of:

[00366] i. incubate the partitioned PCR reactions at a denaturing temperature, which denatures DNA into single-stranded molecules

[00367] ii. incubate the PCR at a very low annealing temperature that allows annealing of both the H-primer and the unaligned part of the L-primer,

[00368] iii. incubate the PCR at the elongation temperature that allows extension of all annealed primers

[00369] iv. optionally, repeat steps I through III, which results in a PCR product.

[00370] 32. The method of item 31, wherein the very low melting temperature is at least 5 °C lower than the low melting temperature.

[00371] 33. The method of any one of items 31 to 32, wherein the very low melting temperature is at least 20 °C lower than the low melting temperature.

[00372] 34. The method of any one of items 31 to 33, wherein the L-primer is an oligonucleotide consisting of:

[00373] • a 5’ sequence of 1 to 10 nucleotides; and

[00374] • a consecutive sequence in the range of 7 to 15 nucleotides that is identical or complementary to a fragment of the target nucleic acid sequence.

[00375] 35. The method of any of the preceding items, wherein the partitioned PCR reactions each contain a detection reagent, which is a variant detection probe, said variant detection probe being capable of hybridizing to the target nucleic acid sequence containing the variant sequence with significantly greater affinity than to the target nucleic acid sequence not containing the variant sequence.

[00376] 36. The method of any of the preceding items, wherein the partitioned PCR reactions each contain a detection reagent that is a wild-type detection probe, said wild-type detection probe being capable of hybridizing to the target nucleic acid sequence that does not contain the variant sequence.

[00377] 37. The method of any one of items 35 and 36, wherein each partitioned PCR reaction contains a variant detection probe and a wild-type detection probe.

[00378] 38. The method of any one of items 35 to 37, wherein the variant detection probe is linked to a fluorophore and a quencher, wherein the quencher is capable of suppressing the fluorescence of the fluorophore, and wherein the fluorophore and the quencher are linked to different nucleotides in the probe.

[00379] 39. The method of any one of items 36 to 38, wherein the wild-type detection probe is linked to a fluorophore and a quencher, wherein the quencher is capable of quenching the fluorescence of the fluorophore, and wherein the fluorophore and the quencher are linked to different nucleotides in the probe.

[00380] 40. The method of any one of items 37 to 39, wherein the variant detection probe is linked to a different fluorophore than the wild-type detection probe.

[00381] 41. The method of any one of items 35 to 40, wherein step g) comprises detecting fluorescence from the fluorophore bound to the variant detection probe.

[00382] 42. The method of any of the preceding items, wherein the H-primer is selected from the group consisting of the H-primer listed in Table 3.

[00383] 43. The method of any of the preceding items, wherein the L-primer is selected from the group consisting of the L-primer listed in Table 3.

[00384] 44. The method of any one of items 35 to 43, wherein the variant detection probe is selected from the group consisting of MUT-Probe listed in Table 3.

[00385] 45. The method of any one of items 36 to 44, wherein the wild-type detection probe is selected from the group consisting of WT-Probe listed in Table 3.

[00386] 46. A method of predicting the presence of a clinical condition in an individual, wherein said clinical condition is linked to the presence of a variant sequence in a target nucleic acid sequence, said method comprising the steps of

[00387] • providing a sample from said individual comprising model nucleic acids

[00388] • perform the method according to any one of items 1 to 45

[00389] wherein the presence of said variant sequence in said target nucleic acid is indicative of the presence of said clinical condition.

[00390] 47. The method according to item 46, wherein the clinical condition is cancer.

[00391] 48. The method according to item 46, wherein the mutation is a mutation associated with cancer.

[00392] 49. The method of any one of items 45 to 48, wherein the sample is a blood sample or a fraction thereof, and the template nucleic acids are selected from the group consisting of cell-free DNA, nucleosome DNA, and circulating tumor DNA.

[00393] 50. The method of any one of items 45 to 49, wherein the sample is selected from the group consisting of saliva samples, urine samples, vaginal fluid samples, ascites fluid samples, cerebrospinal fluid samples, and tissue exudate samples.

[00394] 51. The method of any one of items 45 to 50, wherein the mutation is selected from the group consisting of:

[00395] A. a mutation in PIK3CA, such as any of the H1047R or E542K or E545K mutations;

[00396] B. a mutation in BRAF, such as V600E;

[00397] C. a mutation in KRAS, such as any of the G12D, G12V, G12C, or G13D mutations; and

[00398] D. a mutation in EGFR, such as either the L858R or T790M mutations.

[00399] 52. The method of any one of items 45 to 50, wherein the mutation is selected from the group consisting of the mutations listed in Table 3.

[00400] 53. A method of predicting the presence of a clinical condition in an individual, wherein said clinical condition is linked to the presence of a target nucleic acid sequence, said method comprising the steps of

[00401] • providing a sample from said individual comprising model nucleic acids

[00402] • perform the method according to any one of items 7 to 45

[00403] wherein the presence of said target nucleic acid is indicative of the presence of said clinical condition.

[00404] 54. The method of item 53, wherein the clinical condition is infection with an infectious pathogen.

[00405] 55. The method according to item 54, wherein the target nucleic acid is a nucleic acid sequence from the genome of said pathogen.

[00406] 56. A kit of parts comprising:

[00407] • a primer set comprising at least one pair of primers capable specifically of amplifying a target nucleic acid sequence, wherein the primer set comprises at least one H-primer and one L-primer, wherein the melting temperature of H-primer is at least 10 °C higher than the melting temperature of L-primer, and wherein the L-primer contains a sequence complementary to the elongation product of H-primer,

[00408] • a detection probe is capable of hybridizing to the target nucleic acid sequence, said probe being linked to at least one fluorophore and at least one quencher

[00409] • a nucleic acid polymerase;

[00410] • PCR reagents;

[00411] • reagents for preparing droplets containing partitioned PCR reactions.

[00412] 57. The kit of parts according to item 56, wherein the primer set is as defined in any one of items 10 through 22.

[00413] 58. The kit of parts according to any one of items 56 to 57, wherein the H-initiator is as defined in any one of items 10 to 22.

[00414] 59. The kit of parts according to any one of items 56 to 58, wherein the L-initiator is as defined in any one of items 10 to 22.

[00415] 60. The kit of parts according to any one of items 56 to 59, wherein the detection probe is as defined in any one of items 35 to 45.

[00416] 61. The kit of parts according to any one of items 56 through 60, wherein the H-primer is selected from the group consisting of the H-primer listed in Table 3.

[00417] 62. The kit of parts according to any one of items 56 through 61, wherein the L-primer is selected from the group consisting of the L-primer listed in Table 3.

[00418] 63. The kit of parts according to any one of items 56 through 62, wherein the variant detection probe is selected from the group consisting of the MUT-Probe listed in Table 3.

[00419] 64. The kit of parts according to any one of items 56 to 63, wherein the wild-type detection probe is selected from the group consisting of WT-Probe listed in Table 3.

[00420] 65. The kit of parts according to any one of items 56 through 60, wherein

[00421] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of SEQ ID NO:69 and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of the sequence complementary to SEQ ID NO:69; OR

[00422] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of the complementary sequence to SEQ ID NO:69, and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of SEQ ID NO:69;

[00423] and wherein an H-primer and an L-primer together are capable of amplifying a target sequence comprising at least one of nucleotides 3140, 1624 or 1633 of SEQ ID NO:69.

[00424] 66. The kit of parts according to item 65, wherein the kit of parts comprises a probe capable of detecting:

[00425] • an A to G mutation at nucleotide 3,140 of SEQ ID NO:69;

[00426] • a G to A mutation of nucleotide 1,624 of SEQ ID NO:69; and/or

[00427] • a G to A mutation at nucleotide 1,633 of SEQ ID NO:69.

[00428] 67. The kit of parts according to any one of items 56 through 60, wherein

[00429] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of SEQ ID NO:70 and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of the sequence complementary to SEQ ID NO:70; OR

[00430] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of the complementary sequence to SEQ ID NO:70, and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of SEQ ID NO:70;

[00431] and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising nucleotide 1799 of SEQ ID NO:70.

[00432] 68. The kit of parts according to item 67, wherein the kit of parts comprises a probe capable of detecting a T to A mutation of nucleotides 1,799 of SEQ ID NO:70.

[00433] 69. The kit of parts according to any one of items 56 through 60, wherein

[00434] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of SEQ ID NO:71 and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of the sequence complementary to SEQ ID NO:71; OR

[00435] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of the complementary sequence to SEQ ID NO:71, and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of SEQ ID NO:71;

[00436] and wherein an H-primer and an L-primer together are capable of amplifying a target sequence comprising at least one of nucleotides 2573 or 2369 of SEQ ID NO:71.

[00437] 70. The kit of parts according to item 69, wherein the kit of parts comprises a probe capable of detecting:

[00438] • a T to G mutation of nucleotide 2,573 of SEQ ID NO:71; and/or

[00439] • a C to T mutation at nucleotide 2,369 of SEQ ID NO:71.

[00440] 71. The kit of parts according to any one of items 56 through 60, wherein

[00441] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of SEQ ID NO:72 and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of the sequence complementary to SEQ ID NO:72; OR

[00442] • primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of the complementary sequence to SEQ ID NO:72, and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of SEQ ID NO:72;

[00443] and wherein an H-primer and an L-primer together are capable of amplifying a target sequence comprising at least one nucleotide 34, 35 or 38 of SEQ ID NO:72.

[00444] 72. The kit of parts according to item 71, wherein the kit of parts comprises a probe capable of detecting:

[00445] • a G to A mutation of nucleotide 38 of SEQ ID NO:72.

[00446] • a G to T mutation of nucleotide 34 of SEQ ID NO:72.

[00447] • a G to C mutation of nucleotide 34 of SEQ ID NO:72.

[00448] • a G to A mutation of nucleotide 34 of SEQ ID NO:72.

[00449] • a G to T mutation of nucleotide 35 of SEQ ID NO:72.

[00450] • a G to C mutation of nucleotide 35 of SEQ ID NO:72; and/or

[00451] • a G to A mutation of nucleotide 35 of SEQ ID NO:72.

[00452] 73. The kit of parts according to any one of items 65 to 73, wherein the H-primer consists of said consecutive sequence in the range of 50 to 100 nucleotides, wherein up to 20, such as up to 15, for example, up to 10 nucleotides may have been replaced with a nucleotide analogue, for example, LNA.

[00453] 74. The kit of parts according to any one of items 65 to 73, wherein Primer-H consists of a consecutive sequence in the range of 50 to 100 nucleotides, for example in the range of 50 to 75 nucleotides of said SEQ ID NO.

[00454] 75. The kit of parts according to any one of items 65 to 74, wherein the L-primer consists of said consecutive sequence in the range of 10 to 20 nucleotides and up to 10 additional nucleotides.

[00455] 76. The method of any one of items 1 to 55, wherein the method is carried out using the kit of parts of any one of claims 55 to 75.

EXAMPLES

[00456] The invention is further illustrated by the following Examples, which, however, should not be construed as limiting the invention.

EXAMPLE 1 IBSAFE METHODS (EARLIER INCREMENTAL, THEN SYMMETRICAL, ENHANCED FIDELITY)

[00457] The IBSAFE method is an innovative method to reliably decrease the consequence of any potential DNA polymerase errors so that true positive reactions have a consistent signal advantage over false positive reactions.

[00458] The present example describes the IBSAFE method within a droplet digital PCR setup using target-specific primer pairs and fluorescent probes that discriminate between variant (e.g., mutant) and wild-type (allele) sequences. The method principles can, however, be applied to many different polymerase-based systems.

[00459] The example describes methods for detecting a single nucleotide mutation in a sample that may contain both wild-type and mutant DAN.

[00460] For pilot assays, high Tm primers (H-primer) were designed with a length of ~60 bp and tested to operate relatively efficiently at annealing temperatures of ~72 °C or higher. Probes were designed to overlap the variant base with a length of ~13 to 16 bp, with a balance between positioning the variant base centrally while attempting to maximize Tm. Low Tm primers (L-primer) are designed to be as short as possible with the lowest possible Tm (typically < 48 °C) while achieving adequate sequence specificity and therefore sufficient fluorescent signal quality regardless of fluorophore. For fluorescently labeled quenched probes, typically FAM was used for the mutant allele and HEX for wild-type; but any fluorophore may be used. Probe lengths and Tm's may be similar to that of the L-primer or a few degrees higher. The number of AIPR cycles used can vary, but can typically be 64, and the number of symmetric PCR cycles can vary, but can typically be 27. Since there are many thermal cycles and polymerase loses activity based on the total amount of time at high temperatures, in this example each denaturalization step was chosen to run for 10 seconds (see Table 1 for typical thermal cycling schedule).

[00461] An example of a useful assay design is shown in Figure 2. Figure 2 shows Primer-H and Primer-L, as well as Mutant Detection Probe (MUT Probe) and Wild-Type Detection Probe (WT Probe) for detection of two mutations in the PIK3CA oncogene, namely H1047R and E542K.

[00462] Typical results for IBSAFE compared to a prior art assay are shown in Figure 3. More specifically, Figure 3 shows the result of an IBSAFE assay performed as a droplet digital PCR using the cycling conditions outlined in Table 1, and the primers and probes shown in Figure 2, compared to commercial assays from Bio-Rad Laboratories (PrimePCRTM Mutation Assay PIK3CA p.H1047R, Human, catalog # 100 through 31,246, and PrimePCRTM Mutation Assay PIK3CA WT for p.H1047R, Human, catalog # 100 through 31,249) run according to the manufacturer's standard protocol (hereinafter referred to as the “Commercial Assay”).

[00463] For both commercial and IBSAFE assays, digital droplet PCR was performed using Bio-Rad Laboratories instrumentation including the QX100 Droplet Generator (catalog # 186-3002) and QX100 Droplet Reader (catalog # 186-3001) and QuantaSoft software (catalog # 186-3003). For Bio-Rad Laboratories commercial assays, the manufacturer's protocol was followed. IBSAFE assays were performed according to the methods herein.

[00464] Figure 6 shows the measured mutant allele frequency versus the expected mutant allele frequency. The experiments were performed essentially as described herein above with respect to Figure 3 except that the methods were performed using a template comprising a mixture of wild type and mutant DNA in different ratios as indicated in the figure. While no false positive mutant sequences were detected in the negative controls using the IBSAFE method, the negative control for the commercial assay showed the same amount of mutant (false positive) as a sample with an expected allele frequency of 0.01%. Therefore, when using the IBSAFE method the expected mutant allele frequency is obtained across the tested concentration of mutant template, while the commercial assay results in the same measured mutant allele frequency for samples comprising no mutant DNA, 0.001% and 0.01% mutant DNA. Therefore, the IBSAFE method detects the expected % of mutant alleles even in a sample comprising only 0.001% mutant DNA (see Figure 6 bottom panel). TABLE 1 TYPICAL IBSAFE THERMAL CYCLING CONDITIONS

[00465] Figure 4 shows two different H-Primers, an L-Primer, as well as Mutant Detection Probe (MUT Probe) and Wild-Type Detection Probe (WT Probe) for detecting a mutation in the PIK3CA oncogene, namely H1047R.

[00466] The result of using the primers and probes shown in Figure 4 in different reaction methods is shown in Figure 5. More specifically, partitioned reactions including Primer-H beta 1 or beta 2 along with Primer-L and mutation-specific and wild-type-specific probes as shown in Figure 4 were run with mutation-positive DNA template (not shown; all assays detected the mutation) and wild-type template without AIPR (A), with AIPR at a lower temperature (67°C) (B), and with AIPR at a higher temperature (74°C) (C). Significant reduction of false positive signals is achieved in C.

[00467] IBSAFE and digital droplet PCR were performed as described herein above using the thermocycling conditions outlined in Figure 5.

[00468] Similar IBSAFE reactions were performed using a range of different primers. Figure 5D shows a mutant specific signal and no false positive signals in an assay for detection of the PIK3CA E542K mutation. The following primers and probes were used: *LNA bases indicated by the "+" symbol before the nucleotide

[00469] Figure 5E shows a mutant-specific signal and no false positive signals in an assay for detection of the PIK3CA E545K mutation. The following primers and probes were used: *LNA bases indicated by the "+" symbol before the nucleotide

[00470] The H-primer used for Figures 5D and 5E comprised several LNA bases which resulted in a high melting temperature providing a high Tm difference between the H-primer and the L-primer, such that the AIPR stage was pure asymmetric (one direction) copying of the single-stranded template. As shown no false positives were detected.

[00471] Figure 5F shows a mutant-specific signal and no false positive signals in an assay for detection of the NRAS Q61R mutation. The following primers and probes were used:

[00472] Theoretical calculations assuming 100% efficiency for mutant allele copy numbers per cycle of IBSAFE AIPR and asymmetric PCR versus standard PCR are shown in Table 2. As seen, at a typical point in time for signal detection using the IBSAFE method after cycle 27, the worst-case scenario of introducing a false positive variant in cycle 1 leads to the generation of false nucleic acids at an abundance of approximately 2% compared to the true positive reaction. In contrast, in a standard PCR assay, false positive nucleic acids can be present at approximately 25% of the abundance of true positives in essentially any given cycle (typically, detection is achieved at 40 cycles), contributing to a significant false positive signal. TABLE 2 THEORETICAL CALCULATIONS FOR MUTANT ALLELE COPIES

[00473] The skilled person will be able to design primers and probes useful for detecting other mutations according to the IBSAFE method described herein. Table 3 summarizes non-limiting examples of H-primer, L-primer, and detection probes useful for detecting numerous different mutations.

[00474] Each of the mutations outlined in Table 3 can be detected by performing IBSAFE as described herein, wherein the IBSAFE reaction contains the H-primer, L-primer, WT-Probe, and MUT-Probe indicated for the particular mutation in Table 3. The IBSAFE method can be performed essentially as described in this example, for example, using the thermal cycling conditions outlined in Table 1. The high and low annealing temperatures can be set according to the Tm indicated in Table 3. TABLE 3

EXAMPLE 2

[00475] IBSAFE methods were performed essentially as described in Example 1 except that an extra low temperature PCR step was also used. In this example of an IBSAFE method a misaligned sequence is used for the L-primer. This results in an additional gap in the effective melting temperature difference between Primer-H and Primer-L during the AIPR stage.

[00476] The assay design used in this example is shown in Figure 7A and includes an H-primer, a wild-type detection probe (WT probe), and a variant detection probe (MUT probe). Additionally, the assay includes use of an L-primer comprising 4 misaligned nucleotides. The method used in this example comprises an AIPR step, a low-temperature PCR step followed by an exponential PCR. The exact thermal cycler program used is shown in Figure 7B.

[00477] This example is an IBSAFE assay for the KRAS G13D mutation. A 4-base mismatch sequence is included at the 5’ end of Primer-L and the primer sequence complementary to the target is short. Therefore, Primer-L has a very low melting temperature and will not anneal at the high annealing temperature during the AIPR stage (73°C in this example). In the symmetric stage, the annealing temperature used is very low (30°C in this example). After a few cycles (in this example, 5 cycles), the sequence complementary to the full length Primer-L is incorporated into the synthesized product and the symmetric PCR can be continued at a higher annealing temperature (53°C in this example) where the full length Primer-L is now a perfect match.

[00478] The result is shown in Figure 7B which shows a mutant specific signal and no false positives.

Claims (18)

1. A method for detecting the presence of a target nucleic acid sequence or detecting the presence of a variant sequence in a target nucleic acid sequence in a sample, comprising the steps of a) providing a sample comprising template nucleic acids, b) providing a primer set comprising at least one pair of primers capable specifically of amplifying the target nucleic acid sequence, wherein the primer set comprises at least one H-primer and one L-primer, wherein the melting temperature of the H-primer is at least 16 °C higher, such as at least 20 °C higher, than the melting temperature of the L-primer, and wherein the L-primer contains a sequence complementary to an elongation product fragment of the H-primer, c) providing a nucleic acid polymerase having polymerase activity at an elongation temperature, d) preparing partitioned PCR reactions, each comprising a portion of the sample, the primer set, the nucleic acid polymerase nucleic acid, PCR reagents and, optionally, detection reagents, e) performing an asymmetric incremental polymerase chain reaction (AIPR) comprising the steps of:i. incubating the partitioned PCR reactions at a denaturing temperature, thereby denaturing the DNA into single-stranded molecules,ii. incubating the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer,iii. optionally, incubating the partitioned PCR reactions at the elongation temperature,iv. optionally, repeating steps i to iii,v. thereby amplifying only one strand of the target nucleic acid sequence, f) performing a polymerase chain reaction (PCR) comprising the steps of: 1) incubating the partitioned PCR reactions at a denaturing temperature, thereby denaturing the DNA into single-stranded molecules, 2) incubating the PCR at a low annealing temperature that allows annealing of both H-primer and L-primer, 3) incubating the PCR at the elongation temperature, which allows extension of all annealed primers, 4) optionally repeating steps 1 through 3, 5) thereby amplifying both strands of the target nucleic acid sequence to obtain a PCR product, g) detecting whether the PCR product comprises the target nucleic acid sequence or a variant sequence in the target nucleic acid sequence. 2. The method of claim 1, wherein the variant sequence is a single nucleotide mutation. 3. The method of any one of the preceding claims, wherein the AIPR of step e) comprises the steps of: i. incubating the partitioned PCR reactions at a denaturing temperature, thereby denaturing the DNA into single-stranded molecules, ii. incubating the partitioned PCR reactions at a high annealing temperature that allows annealing of H-primer but not L-primer, wherein the high annealing temperature is also the elongation temperature, which allows extension of the annealed H-primer; iii. repeating steps i to ii, iv. thereby amplifying only one strand of the target nucleic acid sequence. 4. The method of any one of the preceding claims, wherein primer-H is the only primer in the set of primers that has a melting temperature at least 16 °C higher than the melting temperature of primer-L. 5. Method according to any one of the preceding claims, characterized in that the set of primers comprises more than one pair of primers capable of amplifying different target nucleic acid sequences. 6. Method according to any one of the preceding claims, characterized in that the H-primer has a melting temperature at least 16 °C above the melting temperature of any other primer within the primer set that, together with the H-Primer, is capable of amplifying the target nucleic acid sequence. 7. Method according to any one of the preceding claims, characterized in that the high annealing temperature in step e) is at least 10 °C higher, preferably at least 15 °C higher, for example at least 20 °C higher than the melting temperature of initiator-L. 8. The method of any one of the preceding claims, wherein step e) results in elongation of the H-primer but no detectable elongation of any other primer. 9. The method of any one of the preceding claims, wherein the L-primer is a misaligned modified L-primer and the method comprises a low temperature PCR step between steps e) and f), wherein the low temperature PCR comprises the steps of: 1. incubating the partitioned PCR reactions at a denaturation temperature, thereby denaturing the DNA into single-stranded molecules, 2. incubating the PCR at a very low annealing temperature that allows annealing of both the H-primer and the non-misaligned part of the L-primer, 3. incubating the PCR at the elongation temperature, which allows extension of all annealed primers, 4. optionally repeating steps 1) to 3), which results in a PCR product. 10. The method of claim 9, wherein the very low annealing temperature is at least 5 °C lower than the low annealing temperature. 11. The method of any one of claims 9 or 10, wherein the L-primer is an oligonucleotide consisting of: a) a 5' sequence of 1 to 10 nucleotides; and b) a consecutive sequence in the range of 7 to 15 nucleotides that is identical or complementary to a fragment of the target nucleic acid sequence. 12. The method of any preceding claim, wherein the partitioned PCR reactions each contain a detection reagent that is a variant detection probe, said variant detection probe being capable of hybridizing to the target nucleic acid sequence containing the variant sequence with significantly greater affinity than the target nucleic acid sequence not containing the variant sequence, and/or the partitioned PCR reactions each contain a detection reagent that is a wild-type detection probe, said wild-type detection probe being capable of hybridizing to the target nucleic acid sequence not containing the variant sequence. 13. A method of predicting the presence of a clinical condition in an individual, wherein said clinical condition is linked to the presence of a variant sequence in a target nucleic acid sequence or said clinical condition is linked to the presence of a target nucleic acid sequence, said method comprising the steps of a) providing a sample from said individual comprising model nucleic acids, b) performing the method as defined in any one of claims 1 to 12, wherein the presence of said variant sequence in said target nucleic acid or the presence of said target nucleic acid sequence is indicative of the presence of said clinical condition. 14. The method of claim 13, wherein the clinical condition is cancer. 15. A kit of parts when used in the method as defined in any one of claims 1 to 14, comprising: a) a primer set comprising at least one pair of primers capable specifically of amplifying a target nucleic acid sequence, wherein the primer set comprises at least one H-primer and one L-primer, wherein the melting temperature of H-primer is at least 16 °C higher than the melting temperature of L-primer, and wherein L-primer contains a sequence complementary to the elongation product of H-primer, b) a detection probe capable of hybridizing to the target nucleic acid sequence, said probe being linked to at least one fluorophore and at least one quencher, c) a nucleic acid polymerase; d) PCR reagents; e) reagents for preparing droplets containing partitioned PCR reactions, and wherein i) H-primer comprises a consecutive sequence in the range of 50 100 nucleotides of SEQ ID NO:69 and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of the sequence complementary to SEQ ID NO:69; OR ii) primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of the sequence complementary to SEQ ID NO:69, and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of SEQ ID NO:69; and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising at least one of nucleotides 3140, 1624, or 1633 of SEQ ID NO:69. 16. A kit of parts for use in the method as defined in any one of claims 1 to 14, comprising: a) a primer set comprising at least one pair of primers capable specifically of amplifying a target nucleic acid sequence, wherein the primer set comprises at least one H-primer and one L-primer, wherein the melting temperature of H-primer is at least 16 °C higher than the melting temperature of L-primer, and wherein L-primer contains a sequence complementary to the elongation product of H-primer, b) a detection probe capable of hybridizing to the target nucleic acid sequence, said probe being linked to at least one fluorophore and at least one quencher, c) a nucleic acid polymerase; d) PCR reagents; e) reagents for preparing droplets containing partitioned PCR reactions, and wherein i) H-primer comprises a consecutive sequence in the range of 50 to 1000 μg/ml. 100 nucleotides of SEQ ID NO:70 and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of the sequence complementary to SEQ ID NO:70; OR ii) primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of the sequence complementary to SEQ ID NO:70, and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of SEQ ID NO:70; and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising nucleotide 1799 of SEQ ID NO:70. 17. A kit of parts for use in the method as defined in any one of claims 1 to 14, comprising: a) a primer set comprising at least one pair of primers capable specifically of amplifying a target nucleic acid sequence, wherein the primer set comprises at least one H-primer and one L-primer, wherein the melting temperature of H-primer is at least 16 °C higher than the melting temperature of L-primer, and wherein L-primer contains a sequence complementary to the elongation product of H-primer, b) a detection probe capable of hybridizing to the target nucleic acid sequence, said probe being linked to at least one fluorophore and at least one quencher, c) a nucleic acid polymerase; d) PCR reagents; e) reagents for preparing droplets containing partitioned PCR reactions, and wherein i) H-primer comprises a consecutive sequence in the range of 50 to 100 consecutive sequences. 100 nucleotides of SEQ ID NO:71 and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of the sequence complementary to SEQ ID NO:71; OR ii) primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of the sequence complementary to SEQ ID NO:71, and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of SEQ ID NO:71; and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising at least one of nucleotides 2573 or 2369 of SEQ ID NO:71. 18. A kit of parts for use in the method as defined in any one of claims 1 to 14, comprising: a) a primer set comprising at least one pair of primers capable specifically of amplifying a target nucleic acid sequence, wherein the primer set comprises at least one H-primer and one L-primer, wherein the melting temperature of H-primer is at least 16 °C higher than the melting temperature of L-primer, and wherein L-primer contains a sequence complementary to the elongation product of H-primer, b) a detection probe capable of hybridizing to the target nucleic acid sequence, said probe being linked to at least one fluorophore and at least one quencher, c) a nucleic acid polymerase; d) PCR reagents; e) reagents for preparing droplets containing partitioned PCR reactions, and wherein i) H-primer comprises a consecutive sequence in the range of 50 ii) primer-H comprises a consecutive sequence in the range of 50 to 100 nucleotides of the sequence complementary to SEQ ID NO:72, and primer-L comprises a consecutive sequence in the range of 10 to 20 nucleotides of the sequence complementary to SEQ ID NO:72; and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising at least nucleotide 34, 35, or 38 of SEQ ID NO:72.