JP2017163889A - FORWARD PRIMER SET FOR K-ras GENE AMPLIFICATION, KIT FOR K-ras GENE AMPLIFICATION, K-ras GENE AMPLIFICATION METHOD, POLYMORPHISM ANALYSIS METHOD, AND DRUG EFFICACY DETERMINATION METHOD - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a forward primer set with high specificity to nucleic acid in a region including codon 13 of a K-ras gene of wild type and G13D variant for high performance amplification, a kit for K-ras gene amplification, a K-ras gene amplification method, a polymorphism analysis method, and a drug efficacy determination method.SOLUTION: A forward primer of an oligomer comprising a specific sequence, a forward primer set for K-ras gene amplification including the forward primer of the oligomer comprising the specific base sequence, a K-ras gene amplification method which amplifies a region including codon 13 of the K-ras gene using nucleic acid from a sample as a template and the forward primer set for K-ras gene amplification in one reaction solution, and a polymorphism analysis method including polymorphism analysis of codon 13 of the K-ras gene.SELECTED DRAWING: None

Description

  The present invention relates to a forward primer set for K-ras gene amplification, a kit for K-ras gene amplification, a K-ras gene amplification method, a polymorphism analysis method, and a drug efficacy determination method.

  The K-ras gene is a cancer-related gene that is mutated in many malignant tumors. In colorectal cancer, anti-epidermal growth factor receptor (EGFR) antibody drug prediction based on the presence or absence of mutations in the K-ras gene has already been put into practical use. When there is a mutation in the K-ras gene, it is judged that there is a high possibility that the effect of the anti-EGFR antibody drug cannot be expected, and administration of the anti-EGFR antibody drug is not recommended.

  As a mutation of the K-ras gene, it is known as a mutation having particularly many mutations at codons 12 and 13. Among them, in the mutation of codon 13, in the base sequence of the K-ras gene of SEQ ID NO: 6, the guanine (g) of the base of base 224 is replaced with adenine (a), whereby the 13th of the K-Ras protein. In many cases, glycine (G) is mutated to aspartic acid (D) (G13D mutation).

  As a method for detecting mutations in codons 12 and 13 of the K-ras gene, generally, (1) for a target DNA of a sample, a region corresponding to the detection target sequence is amplified by PCR, and the target mutation in the detection target sequence is detected. RFLP (Restriction Fragment Length Polymorphisms) analysis (non-patent document 1) that performs typing by cleaving the amplified product with restriction enzymes that have different cleaving actions depending on the presence or absence of electrophoresis, and (2) detection of the target DNA of the sample A region corresponding to the target sequence is amplified by PCR (Polymerase Chain Reaction), and the entire gene sequence is analyzed (Non-patent Document 2). (3) Allele-specific in which the target mutation is located in the 3 ′ end region -PCR (Allele Specific) in which PCR is performed using specific primers, and mutations are judged by the presence or absence of amplification Primer PCR) method (Non-patent document 3, Non-patent document 4), (4) PCR is carried out using an allele-specific primer in which the target mutation is located in the 3′-terminal region, and mutation is determined by the difference in amplification speed An allyl-specific real-time PCR method (Patent Document 1) to be judged can be mentioned.

  However, these methods require many steps such as purification of DNA extracted from a sample, electrophoresis, treatment with a restriction enzyme and the like, and are troublesome and costly. Moreover, since it is necessary to open the reaction container once after performing PCR, the amplification product may be mixed into the next reaction system, and the analysis accuracy may be reduced. Furthermore, since automation is difficult, it is impossible to analyze a large amount of samples. In the above (3) and (4), a primer in which the target mutation is located in the 3 'end region is used, but there is a problem that sensitivity and specificity are not sufficient.

  In recent years, in order to reduce labor and costs and to automate, a method of analyzing the melting temperature (Tm: melting temperature) of a double-stranded nucleic acid formed from a target nucleic acid and a probe is practical as a point mutation detection method. It has become. Such a method is called, for example, Tm analysis or melting curve analysis. This is the following method. Specifically, first, a hybrid (double-stranded DNA) of the target single-stranded DNA of the detection sample and the probe is formed using a probe complementary to the detection target sequence containing the point mutation for detection. Subsequently, the hybrid formed body is subjected to a heat treatment, and the dissociation (melting) of the hybrid accompanying a temperature rise is detected by a change in signal such as absorbance. And it is the method of judging the presence or absence of a point mutation by determining Tm value based on this detection result. The Tm value is higher as the homology of the hybrid is higher, and lower as the homology is lower. For this reason, a Tm value (evaluation reference value) is previously obtained for a hybrid of a detection target sequence containing a point mutation and a probe complementary thereto, and the Tm value of the target single-stranded DNA of the detection sample and the probe (Measurement value) is measured, and if the obtained measurement value is the same as the evaluation reference value, it can be determined that there is a match, that is, a point mutation exists in the target DNA. On the other hand, if the measured value is lower than the evaluation reference value, it can be determined that there is a mismatch, that is, there is no point mutation in the target DNA. And according to this method, automation is also possible (patent documents 2 and 3).

In addition, in order to improve the specificity of the allele-specific primer, methods such as introducing a mismatch base at a position about the third base from the 3 ′ end of the primer, raising the annealing temperature, etc. can be taken. A method for increasing specificity usually reduces nucleic acid amplification efficiency. If the nucleic acid amplification efficiency is low, a sufficient amount of amplification product cannot be obtained, so that the detection sensitivity of the target gene decreases.
When detecting a G13D mutant K-ras gene, the copy number of the G13D mutant K-ras gene in the test sample should be sufficiently larger than the copy number of the K-ras gene whose codon 13 is wild type. For example, a correct determination may be made even when such a primer with low specificity is used.
However, when the copy number of the G13D mutant K-ras gene derived from a tumor or the like is significantly smaller than the copy number of the wild-type K-ras gene derived from a normal tissue, the wild type having a large copy number A primer specific for the G13D mutant allele binds to the K-ras gene of the nuclease, and nonspecific nucleic acid amplification is easily performed. When the copy number of the G13D mutant K-ras gene in the test sample is remarkably smaller than the copy number of the wild-type K-ras gene of codon 13, for example, normal tissue is added to the tumor tissue as the test sample. Or when using circulating DNA as a test sample.
On the other hand, if an attempt is made to suppress an amplification reaction that is not specific to the G13D mutant allele, nucleic acid amplification of the G13D mutant allele that does not have a sufficient copy number is not performed efficiently, and false negatives are likely to occur.

  As described above, in the detection of a mutant form of the K-ras gene, it is possible to automate with reduced labor and cost, and a technique having high detection sensitivity and specificity has been demanded.

International Publication No. 2011/104695 Japanese Patent No. 3963422 International Publication No. 2011/071046

American Journal of Pathology (1993) Aug; 143, pp 545-554 Yonsei Med J. (2009) Apr; 50 (2), pp 266-72 Japan Gastroenterological Surgery Journal (1997); 30 (4), pp 897-900 Japanese Journal of Colorectal Anal Diseases (1997); 50, pp 33-40

In order to increase the detection sensitivity of the G13D mutant K-ras gene, a method of amplifying the nucleic acid and specifically amplifying the region including the mutant type and wild type codon 13 can be considered. When such an allele-specific nucleic acid amplification is performed, it is possible to specifically amplify each allele of the wild type and the G13D mutant and to perform the amplification efficiently. It greatly contributes to the improvement of sensitivity and specificity. As described above, when allyl-specific nucleic acid amplification is performed, compatibility between allyl specificity and amplification efficiency is a problem.
In particular, when it is necessary to perform nucleic acid amplification with high efficiency in order to obtain high detection sensitivity, it is more difficult because both the allyl specificity and the amplification efficiency are required at a high level.

  The present invention provides a forward primer set for amplifying a nucleic acid in the region containing codon 13 of the K-ras gene with high specificity and high efficiency for each of the wild type and G13D mutants, a kit for K-ras gene amplification, An object is to provide a ras gene amplification method, a polymorphism analysis method, and a drug efficacy determination method.

Means for solving the above problems are as follows.
<1> K-ras gene amplification forward primer set for amplifying a region containing codon 13 of the K-ras gene by the nucleic acid amplification method, which is a forward primer set of the following (1), (2) or (3) :
(1) a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 1, a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 3,
Forward primer set, including
(2) a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 2, a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 4,
Forward primer set, including
(3) a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 2, a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 5,
Forward primer set.
<2> A K-ras gene amplification method using the K-ras gene amplification forward primer set according to <1>.
<3> Amplifying a region containing codon 13 of the K-ras gene using the nucleic acid in the sample as a template and the K-ras gene amplification forward primer set in one reaction solution, The K-ras gene amplification method described.
<4> amplifying a region containing codon 13 of the K-ras gene by the K-ras gene amplification method according to <2> or <3>;
Forming a hybrid between a single-stranded nucleic acid obtained from the amplification product of the amplification and a probe hybridizing to the single-stranded nucleic acid obtained from the amplification product;
Changing the temperature of the reaction solution containing the hybrid, and measuring the signal variation based on the dissociation state of the hybrid;
Analyzing a polymorphism of codon 13 of the K-ras gene based on the variation of the signal.
<5> The polymorphism analysis method according to <4>, wherein the amplification and the formation of the hybrid proceed simultaneously.
<6> The polymorphism analysis method according to <4> or <5>, wherein the probe is a probe that hybridizes to a region containing codon 13 of the K-ras gene.
<7> The polymorphism analysis method according to any one of <4> to <6>, wherein the probe is a fluorescently labeled probe.
<8> analyzing the polymorphism of codon 13 of the K-ras gene by the polymorphism analysis method according to any one of <4> to <7>;
Determining the efficacy of the drug based on the analysis result obtained by the analysis.
<9> The drug efficacy determination method according to <8>, wherein the drug is an anti-epidermal growth factor receptor antibody drug.
<10> A kit for amplifying a K-ras gene for amplifying a region containing the codon 13 of the K-ras gene, comprising the forward primer set for amplifying the K-ras gene according to <1>.
<11> The kit for amplifying a K-ras gene according to <10>, comprising one or more reverse primers for amplifying a region containing codon 13 of the K-ras gene.
<12> The kit for amplifying a K-ras gene according to <10> or <11>, comprising a probe that hybridizes to a region containing codon 13 of the K-ras gene.
<13> The kit for amplifying a K-ras gene according to <12>, wherein the probe is a fluorescently labeled probe.

  According to the present invention, a forward primer set for amplifying a region containing codon 13 of the K-ras gene by a nucleic acid amplification method, a kit for K-ras gene amplification, a K-ras gene amplification method, a polymorphism analysis method, and A medicinal effect determination method can be provided.

(A) is an example of a melting curve of a nucleic acid mixture, and (B) is an example of a differential melting curve of a nucleic acid mixture. It is a differential melting curve obtained by Tm value analysis when nucleic acid amplification was performed using a template containing a wild type K-ras gene. A differential melting curve obtained by Tm value analysis when nucleic acid amplification was performed using a template containing a G13D mutant K-ras gene so that the copy number was 0.3% with respect to the wild-type K-ras gene. is there.

  Hereinafter, modes for carrying out the present invention will be described. However, the present invention is not limited to the following embodiments.

In the present specification, a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In addition, when referring to the amount of a certain component in the composition in the present specification, when there are a plurality of substances corresponding to the component in the composition, the amount is determined in the composition unless otherwise defined. Means the total amount of the plurality of substances present.
In addition, in this specification, the term “process” is not limited to an independent process, and even if it cannot be clearly distinguished from other processes, the term “process” is used if the intended purpose of the process is achieved. included.
Further, in this specification, the “Tm value” is a temperature at which a double-stranded nucleic acid is dissociated (melting temperature: Tm), and generally a temperature at which the absorbance at 260 nm reaches 50% of the total increase in absorbance. It is defined as When a solution containing a double-stranded nucleic acid, such as a double-stranded DNA, is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both strands in double-stranded DNA are unwound by heating and dissociated into single-stranded DNA (DNA melting). When all the double-stranded DNAs are dissociated into single-stranded DNAs, the absorbance is about 1.5 times the absorbance at the start of heating (absorbance of only the double-stranded DNA), thereby dissociating. It can be judged that it has been completed. The Tm value is set based on this phenomenon.

<Forward primer set for K-ras gene amplification>
A forward primer set for K-ras gene amplification according to an embodiment of the present invention (hereinafter simply referred to as “forward primer set of the present invention”) is the following forward primer set (1), (2) or (3). Yes, it is used to amplify the region containing codon 13 of the K-ras gene by the nucleic acid amplification method.
Forward primer set (1)
A forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 1,
A forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 3,
Forward primer set, including
Forward primer set (2)
A forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 2,
A forward primer which is an oligonucleotide consisting of the base sequence of SEQ ID NO: 4,
Forward primer set, including
Forward primer set (3)
A forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 2,
A forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 5,
Forward primer set.

As will be described in detail later, the forward primer sets (1) to (3) are respectively a forward primer for specifically amplifying a region containing the wild-type codon 13 of the K-ras gene, and the K-ras gene. And a forward primer for specifically amplifying a region containing codon 13 of the G13D variant.
According to such a forward primer set of the present invention, the region containing codon 13 of the K-ras gene can be amplified with high specificity and high efficiency for each of the wild type and the mutant type.

In the forward primer sets (1) to (3), the forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and the forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 2 are each of the K-ras gene. It is a forward primer for specifically amplifying a region containing wild-type codon 13.
A forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 3, a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 4, and a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 5, respectively, are K-ras. It is a forward primer for specifically amplifying a region containing the codon 13 of the G13D variant of the gene.

  In the K-ras gene, the base sequence of wild-type codon 13 is GGC and encodes glycine (G). The base sequence of codon 13 of the G13D variant is GAC and encodes aspartic acid (D).

The base sequences shown as SEQ ID NO: 1 to SEQ ID NO: 5 are described below.
-SEQ ID NO: 1 CTAGCtagttggagctggtgG
-SEQ ID NO: 2: CTAGCagttggagctggtgG
-SEQ ID NO: 3: TGCTCtggtagttggagctggtgA
-SEQ ID NO: 4: TGCTCggtagttggagctggtgA
-SEQ ID NO: 5: TGCTCgtagttggagctggtgA
In SEQ ID NO: 1 to SEQ ID NO: 5, the base indicated by capital letters at the 3 ′ end is a base corresponding to the polymorphic site of codon 13 of the K-ras gene. The base indicated by capital letters at the 5 ′ end is an artificially added sequence that does not have complementarity in the binding region of the K-ras gene of each primer.

The forward primer which is an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and the forward primer which is an oligonucleotide consisting of the base sequence of SEQ ID NO: 2 have a guanine (G) at the 3 ′ terminal base, and the codon 13 wild type It has complementarity with the polymorphic site of the antisense strand of the K-ras gene.
The forward primer which is an oligonucleotide consisting of the base sequence of SEQ ID NO: 3, the forward primer which is an oligonucleotide consisting of the base sequence of SEQ ID NO: 4 and the forward primer which is an oligonucleotide consisting of the base sequence of SEQ ID NO: 5 are each at the 3 ′ end This base is adenine (A) and has a complementarity with the polymorphic site of the antisense strand of the G13D mutant K-ras gene.

The base sequence of the K-ras gene is, for example, GenBank Accession No. In NG_007524, it is registered as the 5001st to 50675th area. SEQ ID NO: 6 is a partial sequence of the K-ras gene, and corresponds to the 10351st to 10850th region in the base sequence of the accession number. The sequence of the codon 13 corresponds to the region 10573 to 10575 in the base sequence of the accession number.
The forward primer set of the present invention is preferably used when a region containing codon 13 of the K-ras gene in a biological sample such as a tissue sample or a whole blood sample is amplified.

<K-ras gene amplification method>
The K-ras gene amplification method according to an embodiment of the present invention (hereinafter simply referred to as “the K-ras gene amplification method of the present invention”) uses the nucleic acid in a sample as a template as the forward primer set (1) to Using any one of (3), the region containing codon 13 of the K-ras gene is specifically amplified to each of the wild type and G13D mutant types. According to the K-ras gene amplification method, it is possible to amplify a region containing the codon 13 of the K-ras gene with high specificity and high efficiency for each of the wild-type codon 13 or the G13D mutant codon 13. . In the K-ras gene amplification method of the present invention, nucleic acid amplification can also be performed using the forward primer set of the present invention in one reaction solution.

  Examples of the sample containing the template nucleic acid include a biological sample. Biological samples include whole blood, oral cells such as oral mucosa, somatic cells such as nails and hair, germ cells, sputum, amniotic fluid, paraffin-embedded tissue, urine, gastric juice, gastric lavage fluid, or suspensions thereof. Is mentioned. Further, a nucleic acid isolated from a biological sample may be used as a template. For example, a commercially available genomic DNA isolation kit can be used for isolation of genomic DNA from whole blood. Alternatively, an amplification product obtained by amplifying DNA contained in a biological sample by a gene amplification method may be used as a template. Alternatively, an amplification product obtained by generating cDNA from RNA contained in a biological sample by a reverse transcription PCR reaction and amplifying the cDNA by a gene amplification method may be used as a template.

  The nucleic acid amplification method is not particularly limited. Examples of the nucleic acid amplification method include a PCR method, a NASBA (Nucleic Acid Sequence Based Amplification) method, a TMA (Transcription-Mediated Amplification) method, an SDA (Strand Displacement Amplification) method, and the PCR method is preferred.

  In the amplification, the sample addition ratio in the amplification reaction solution is not particularly limited. As a specific example, when the sample is a biological sample (for example, a whole blood sample), the lower limit of the addition ratio is preferably 0.01% by volume or more, more preferably 0.05% by volume or more, More preferably, it is 0.1 volume% or more. When the sample is a biological sample (for example, a whole blood sample), the upper limit of the addition ratio is preferably 2% by volume or less, more preferably 1% by volume or less, and 0.5% by volume or less. More preferably.

  In addition, in the polymorphism analysis method described later, for example, when optical detection using a labeled probe is performed, the addition ratio of the biological sample in the reaction solution is set to, for example, 0.1 volume% to 0.5 volume% It is preferable to do. If it is this range, the influence by generation | occurrence | production of the precipitate by denaturation etc. can fully be prevented, for example, and the measurement precision by an optical method can be improved. In addition, since PCR inhibition by contaminants in the biological sample is sufficiently suppressed, it is expected that the amplification efficiency can be further improved.

Moreover, it is preferable to further add albumin to the reaction solution before starting the amplification reaction. By adding such albumin, for example, the influence due to the occurrence of precipitates or turbidity can be further reduced, and the amplification efficiency is further improved.
The addition ratio of albumin in the reaction solution is, for example, 0.01% by mass to 2% by mass, preferably 0.1% by mass to 1% by mass, and more preferably 0.2% by mass to 0.8% by mass. % By mass. Examples of albumin include bovine serum albumin (BSA), human serum albumin, rat serum albumin, horse serum albumin and the like, and are not particularly limited. Any one of these albumins may be used, or two or more of them may be used in combination.

  Hereinafter, amplification will be described by taking the PCR method as an example, but the present invention is not limited to this example.

First, a PCR reaction solution containing a template nucleic acid, a forward primer set of the present invention, and a reverse primer that anneals to a region downstream of codon 13 of the K-ras gene is prepared.
The addition ratio of various primers in the PCR reaction solution is not particularly limited. For example, when the primer set (1), (2) or (3) is used, the total addition ratio of the two forward primers and the reverse primer is preferably 0.01 μmol / L to 50 μmol / L, More preferably, it is 0.5 μmol / L to 5 μmol / L.
Moreover, the total addition ratio of the two forward primers is preferably 0.01 μmol / L to 10 μmol / L, and more preferably 0.1 μmol / L to 1 μmol / L. The addition ratio of the reverse primer is preferably 0.05 μmol / L to 50 μmol / L, and more preferably 0.5 μmol / L to 5 μmol / L.

  The content ratio of the forward primer set and the reverse primer in the PCR reaction solution is not particularly limited. From the viewpoint of increasing the amplification efficiency and specificity to the wild type or mutant type, the ratio between the total molar amount of the forward primer set (1), (2) or (3) and the total molar amount of the reverse primer is: The ratio is preferably 1: 1 to 1:10, more preferably 1: 2 to 1: 5, and still more preferably 1: 3 to 1: 4.5.

  Further, a forward primer for specifically amplifying a region containing the wild-type codon 13 of the K-ras gene contained in the forward primer set, and a region containing the codon 13 of the G13D mutant type of the K-ras gene are specified. The content ratio in the PCR reaction solution with the forward primer for amplification is not particularly limited. From the viewpoint of increasing the amplification efficiency and specificity, it is preferably 0.5: 2 to 1: 0.5, and more preferably 0.8 to 1.2: 1.2 to 0.8. Particularly preferred is 1: 1.

  Other composition components in the PCR reaction solution are not particularly limited, and include conventionally known components, and the ratio thereof is not particularly limited. Examples of other composition components include nucleotides such as DNA polymerase and nucleoside triphosphate (dNTP), solvents, and the like. In the PCR reaction solution, the order of adding each composition component is not limited.

The DNA polymerase is not particularly limited. For example, a conventionally known heat-resistant bacterium-derived polymerase can be used. Specific examples include DNA polymerase derived from Thermus aquaticus (see US Pat. Nos. 4,889,818 and 5,079,352) (Taq polymerase (trade name)), Thermus thermophilus (Thermus thermophilus). ) -Derived DNA polymerase (see WO91 / 09950) (rTth DNA polymerase), Pyrococcus furiosus-derived DNA polymerase (see WO92 / 96889) (Pfu DNA polymerase; manufactured by Strategene) ), DNA polymerase derived from Thermococcus litoralis (see European Patent No. 0455430) (Vent (trademark); manufactured by New England Biolabs) and the like are commercially available. Of these, thermostable DNA polymerase derived from Thermus aquaticus is preferred.
The addition ratio of DNA polymerase in the PCR reaction solution may be a ratio that is usually used in the art for the purpose of amplifying the target nucleic acid.

Examples of nucleoside triphosphates generally include dNTPs (eg, dATP, dGTP, dCTP, dTTP, dUTP, etc.). The addition ratio of dNTP in the PCR reaction solution may be a ratio that is usually used in the art for the purpose of amplifying the target nucleic acid.
As the solvent, Tris-HCl, Tricine, MES (2-morpholinoethanesulfonic acid), MOPS (3-morpholinopropanesulfonic acid), HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), CAPS (N-cyclohexyl-3-) For example, a buffer solution such as aminopropanesulfonic acid may be used, and a commercially available buffer solution for PCR, a buffer solution attached to the PCR kit, or the like may be used as it is.
The PCR reaction solution may contain glycerol, heparin, betaine, NaN ₃ , KCl, MgCl ₂ , MgSO _{4 and the} like.

PCR usually involves (i) dissociation of double-stranded nucleic acid into single-stranded nucleic acid (dissociation step), (ii) annealing of primer to template nucleic acid (annealing step), and (iii) nucleic acid from primer by DNA polymerase. It includes 3 steps of sequence extension (extension step). The conditions for each step are not particularly limited. The conditions for the dissociation step are preferably, for example, 90 ° C. to 99 ° C. and 1 second to 120 seconds, and more preferably 92 ° C. to 95 ° C. and 1 second to 60 seconds. The conditions for the annealing step are, for example, preferably 40 ° C. to 70 ° C. and 1 second to 300 seconds, more preferably 50 ° C. to 70 ° C. and 5 seconds to 60 seconds. In addition, the conditions for the elongation step are preferably, for example, 50 ° C. to 80 ° C., 1 second to 300 seconds, and more preferably 50 ° C. to 80 ° C., 5 seconds to 60 seconds. The number of cycles is not particularly limited. For example, 30 cycles or more are preferable with 3 steps as 1 cycle. The upper limit is not particularly limited.
For example, the total is 100 cycles or less, preferably 70 cycles or less, more preferably 50 cycles or less. What is necessary is just to control automatically the temperature change of each process using a thermal cycler etc., for example. The annealing process and the extension process may be performed under the same temperature condition, and PCR may be performed in two processes.

  As described above, an amplification product in which the region containing the wild-type codon 13 of the K-ras gene and / or the G13D mutant-type codon 13 is allele-specifically amplified can be obtained.

-Reverse primer-
The reverse primer used for nucleic acid amplification is not particularly limited, and can be set by a conventionally known method. A person skilled in the art can obtain a reverse primer capable of amplifying the nucleic acid in the region containing codon 13 of the K-ras gene in combination with the forward primer set according to one embodiment of the present invention.
The reverse primer is preferably set to a Tm value close to that of the forward primer included in the forward primer set of the present invention. For example, the difference from the Tm value of the forward primer is preferably set within 10 ° C, more preferably within 5 ° C. The calculation of the Tm value can be performed as described later. Such a primer design method is known to those skilled in the art. For example, design tools such as Oligo Analyzer (http://sg.idtdna.com/calc/analyzer) are publicly available, and these can also be used.
The base length of the amplification product is not particularly limited, and can be set to, for example, 50 mer to 1000 mer, or 80 mer to 200 mer.

As a reverse primer capable of amplifying the nucleic acid in the region containing codon 13 of the K-ras gene in combination with the forward primer set of the present invention, specifically, an oligonucleotide comprising the base sequence shown in SEQ ID NO: 7 shown below However, the present invention is not limited to this.
-SEQ ID NO: 7: ggtcctgcaccagtaatatgca

  The reverse primer specifically includes the forward primer for specifically amplifying the region containing the wild type codon 13 of the K-ras gene and the region containing the codon 13 of the G13D mutant, which are included in the forward primer set of the present invention. Two kinds of reverse primers combined with each of the forward primers for amplification may be used. However, from the viewpoint of efficiency, it is combined with both the forward primer for specifically amplifying the region containing the wild type codon 13 and the forward primer for specifically amplifying the region containing the codon 13 of the G13D mutant type. It is preferable to use one kind of common primer that can be used.

<Polymorphism analysis method>
The polymorphism analysis method according to an embodiment of the present invention (hereinafter simply referred to as “polymorphism analysis method of the present invention”) is a region containing codon 13 of the K-ras gene by the K-ras gene amplification method of the present invention. And a single-stranded amplified nucleic acid obtained by the amplification step and a probe capable of hybridizing to a region containing the polymorphic site of codon 13 of the K-ras gene. A hybridization step, a signal measurement step of measuring a change in signal based on the dissociation state of the hybrid by changing the temperature of the reaction solution containing the hybrid, and a codon 13 of the K-ras gene based on the change in the signal. An analysis step for analyzing the polymorphism. According to the polymorphism analysis method of the present invention, using the nucleic acid in the sample as a template, the codon 13 gene polymorphic site of the K-ras gene is a wild-type nucleic acid and the codon 13 gene polymorphic site of the K-ras gene A nucleic acid having a G13D mutant can be simultaneously amplified in the same reaction solution with high efficiency and high specificity to the wild type or the G13D mutant, respectively, and the polymorphism of codon 13 of the K-ras gene can be analyzed.

  Since the nucleic acid amplification is the same as the nucleic acid amplification in the K-ras gene amplification method of the present invention, detailed description thereof is omitted.

  Next, in the hybridization step, a probe that can hybridize to the region containing the polymorphic site of codon 13 of the K-ras gene and the single-stranded amplified nucleic acid obtained in the amplification step (polymorphism analysis) For example). The single-stranded amplified nucleic acid can be prepared, for example, by heating the reaction solution and dissociating the double-stranded amplified nucleic acid that is the amplification product obtained in the amplification step. Details of the polymorphism analysis probe will be described later.

The timing at which the polymorphism analysis probe is added to the reaction solution is not particularly limited. For example, it may be before the amplification process, at the start of the amplification process, in the middle of the amplification process, or after the amplification process. Of these, addition before the amplification step or at the start of the amplification step is preferable because the amplification reaction and the hybridization can be performed continuously. That is, it is preferable from the viewpoint of processing efficiency that the amplification step and the hybridization step proceed simultaneously.
The addition ratio of the polymorphism analysis probe in the reaction solution is not particularly limited. For example, the polymorphism analysis probe is preferably added so as to be in the range of 10 nmol / L to 400 nmol / L, and more preferably added so as to be in the range of 20 nmol / L to 200 nmol / L.

There is no particular limitation on the hybridization technique and conditions of the single-stranded amplified nucleic acid and the polymorphism analysis probe. Conditions known in the art may be applied as they are for the purpose of dissociating double-stranded amplified nucleic acids into single-stranded amplified nucleic acids and hybridizing single-stranded nucleic acids.
For example, the heating temperature in the dissociation is not particularly limited as long as the amplification product can be dissociated, but is, for example, 85 ° C to 95 ° C. The heating time is not particularly limited, but is usually 1 second to 10 minutes, preferably 1 second to 5 minutes. In addition, hybridization between the dissociated single-stranded amplified nucleic acid and the polymorphism analysis probe can be performed, for example, by lowering the heating temperature in dissociation after dissociation. The temperature condition is, for example, 40 ° C to 50 ° C.

  In the amplification step, when the forward primer set of the present invention and the polymorphism analysis probe coexist, the polymorphism analysis probe prevents the polymorphism analysis probe itself from becoming a reaction target of DNA polymerase. Therefore, it is preferable that a fluorescent label described later is added to the 3 ′ end side of the polymorphism analysis probe or a phosphate group is added.

  In addition, the polymorphism analysis probe is preferably a labeled probe with a label, from the viewpoint of detection efficiency. For example, when two types of probes specific to the amplified product of the wild type codon 13 and the amplified product of the codon 13 of the G13D mutant type are used, they are labeled with different labels that are detected under different conditions. It is preferable. By using different labels in this way, each amplification product can be analyzed separately by changing detection conditions even in the same reaction solution.

  Specific examples of the labeling substance in the labeling probe include a fluorescent dye and a fluorophore. As the labeled probe, it is preferable to use a fluorescent labeled probe labeled with such a fluorescent dye or fluorophore. As a specific example of a fluorescently labeled probe, it is labeled with a fluorescent dye, exhibits fluorescence alone (when not hybridized to a complementary sequence), and decreases when hybridized (when hybridized to a complementary sequence). (For example, a quenching probe).

A probe that uses such a quenching phenomenon is generally called a fluorescence quenching probe. In the fluorescence quenching probe, the base of the 3 ′ region (eg, 3 ′ end) or 5 ′ region (eg, 5 ′ end) of the oligonucleotide is preferably labeled with a fluorescent dye. Is preferably cytosine (C). In this case, in the detection target sequence to which the fluorescence quenching probe hybridizes, the base paired with the terminal base C of the fluorescence quenching probe or the base separated by 1 to 3 bases from the paired base becomes guanine (G). It is preferable to design the base sequence of the fluorescence quenching probe. Such a fluorescence quenching probe is generally called a guanine quenching probe and is known as a so-called Q Probe (registered trademark).
When such a guanine quenching probe is hybridized to the detection target sequence, the cytosine (C) at the end labeled with the fluorescent dye approaches guanine (G) in the detection target sequence, so that the emission of the fluorescent dye becomes weak ( This shows the phenomenon that the fluorescence intensity decreases. By using such a guanine quenching probe, hybridization and dissociation can be easily confirmed by signal fluctuation. The labeling substance can usually be bound to a phosphate group of a nucleotide.

  In addition to the detection method using Q Probe, a known detection mode may be applied. Examples of such detection modes include Taq-man Probe method, RFLP method, Hybridization Probe method, Molecular Beacon method, MGB probe method and the like.

  The fluorescent dye is not particularly limited, and examples thereof include fluorescein, phosphor, rhodamine, and polymethine dye derivatives. Examples of commercially available fluorescent dyes include Pacific Blue (registered trademark, manufactured by Molecular Probes), TAMRA (registered trademark, manufactured by Molecular Probes), BODIPY FL (registered trademark, manufactured by Molecular Probes), FluorePrime (trade name, Amersham). Pharmacia), Cy3 and Cy5 (trade name, manufactured by Amersham Pharmacia), Fluoredite (trade name, manufactured by Millipore), FAM (registered trademark, manufactured by ABI), and the like. The combination of fluorescent dyes used for a plurality of probes is not particularly limited as long as it can be detected under different conditions. For example, Pacific Blue (detection wavelength: 450 nm to 480 nm), TAMRA (detection wavelength: 585 nm to 700 nm), and BODIPY FL (Detection wavelength: 515 nm to 555 nm) and the like.

  In addition, when the polymorphism analysis probe is a labeled probe labeled with a labeling substance such as a fluorescent dye, an unlabeled probe having the same sequence as the labeled probe may be used in combination. Thereby, for example, the advantage that signal intensity, such as fluorescence intensity to detect, can be adjusted is acquired. This unlabeled probe may have a phosphate group added to its 3 'end.

  Next, in the signal measurement step, the temperature of the reaction solution containing the hybrid is changed, and the fluctuation of the signal based on the dissociation state of the hybrid is measured. The measurement of the signal indicating the dissociation state of the hybrid may be an absorbance measurement at 260 nm, but is preferably a signal measurement of a labeling substance. Detection sensitivity can be increased by measuring the signal of the labeling substance.

  The fluctuation of the signal based on the dissociation state of the hybrid is performed by changing the temperature of the reaction solution. For example, the reaction solution is heated, that is, the hybrid of the single-stranded amplified nucleic acid and the polymorphism analysis probe is heated, and the fluctuation of the signal accompanying the temperature rise is measured. As described above, for example, when a labeled probe (guanine quenching probe) in which the terminal C base is labeled is used, the fluorescence decreases (or quenches) when hybridized with the single-stranded amplified nucleic acid, and dissociation occurs. Fluorescence is emitted in this state. Therefore, for example, a hybrid in which fluorescence is decreased (or quenched) may be gradually heated to measure an increase in fluorescence intensity as the temperature rises. When a labeled probe is used, the signal can be measured under conditions according to the labeling substance of the labeled probe.

  The temperature range for measuring the signal fluctuation is not particularly limited. For example, the start temperature is room temperature to 85 ° C, preferably 25 ° C to 70 ° C, and the end temperature is 40 ° C to 105 ° C. Further, the rate of temperature rise is not particularly limited, but is, for example, 0.1 ° C./second to 20 ° C./second, preferably 0.3 ° C./second to 5 ° C./second.

Next, in the analysis step, the polymorphism of codon 13 of the K-ras gene is analyzed based on the change in the signal. In that case, it is preferable to analyze the fluctuation | variation of the signal obtained at the said signal measurement process, determine Tm value, and to analyze the polymorphism of the codon 13 of a K-ras gene based on this Tm value.
The determination of the Tm value can be performed as follows, for example. As described above, for example, when a labeled probe labeled with a terminal C base (guanine quenching probe) is used, the amount of change in fluorescence intensity per unit time at each temperature can be calculated from the variation in fluorescence intensity obtained. calculate. When the change amount is (−d (fluorescence intensity increase amount) / dt), for example, the temperature showing the lowest value can be determined as the Tm value. When the amount of change is (d (fluorescence intensity increase amount) / dt), for example, the temperature showing the highest value can be determined as the Tm value.
When a probe that does not show a signal alone and shows a signal by hybridization is used as a labeled probe, on the contrary, the decrease in fluorescence intensity may be measured.

In the analysis step, the polymorphism of codon 13 of the K-ras gene can be analyzed based on the Tm value thus determined.
For example, when detecting the codon 13 of the G13D mutant type of the K-ras gene, a probe for polymorphism analysis that is completely complementary to the detection target sequence containing the mutant base is used, and the Tm value of the formed hybrid is If the Tm value of a completely complementary hybrid determined in advance as a reference value is the same, the target base can be determined as a G13D mutant. In addition, if the Tm value of the formed hybrid is the same as the Tm value of a hybrid different by one base that has been determined in advance as a reference value (a value lower than the Tm value of a completely complementary hybrid), the target base is It can be judged as a wild type. When both Tm values are detected, it can be determined that the nucleic acid showing the G13D mutant and the nucleic acid showing the wild type coexist.

  In the above description, the hybrid is heated in the signal measurement step, and the signal fluctuation accompanying the temperature rise is measured. However, the fluctuation of the signal at the time of hybridization may be measured. That is, when a hybrid is formed by lowering the temperature of the reaction solution containing the polymorphism analysis probe, signal fluctuations accompanying the temperature drop may be measured.

  As a specific example, when a labeled probe (for example, a guanine quenching probe) that shows a signal alone and does not show a signal due to hybridization is used, fluorescence is emitted when the single-stranded amplified nucleic acid and the labeled probe are dissociated. However, when a hybrid is formed by a decrease in temperature, the fluorescence is reduced (or quenched). Therefore, for example, the temperature of the reaction solution may be gradually lowered to measure the decrease in fluorescence intensity accompanying the temperature drop. On the other hand, when a labeled probe that does not show a signal alone and shows a signal by hybridization, it does not emit fluorescence when the single-stranded amplified nucleic acid and the labeled probe are dissociated. When a hybrid is formed, it becomes fluorescent. Therefore, for example, the temperature of the reaction solution may be gradually lowered and the increase in fluorescence intensity associated with the temperature drop may be measured.

  In order to determine the genotype of codon 13 when the nucleic acid sequences of the wild-type codon 13 of the K-ras gene and the G13D mutant codon 13 are mixed, the relationship between the temperature and the differential value of the signal intensity is used. It can also be performed by calculating the peak areas of the Tm values of the wild type codon 13 and the G13D mutant codon 13 in the melting curve represented (also referred to as a differential melting curve).

First, for example, for a sample containing a nucleic acid mixture of a wild-type nucleic acid Wt and a G13D mutant nucleic acid Mt, a melting curve is obtained using a melting curve analyzer.
FIG. 1A shows a melting curve represented by the relationship between the temperature of a certain nucleic acid mixture and a detection signal such as absorbance or fluorescence intensity, and FIG. 1B shows a differential melting curve. By detecting the peak from this differential melting curve, the melting temperature TmW of the nucleic acid Wt and the melting temperature TmM of the nucleic acid Mt are detected, and each temperature range including TmW and TmM is set.

As the temperature range ΔTW including TmW, for example, a temperature range is set such that the temperature at which the differential value of the detection signal is minimum between TmW and TmM is the lower limit and the temperature corresponding to the base of the detection signal is the upper limit. be able to. Further, as the temperature range ΔTM including TmM, for example, a temperature range in which the temperature at which the differential value of the detection signal is minimum between TmW and TmM is the upper limit and the temperature corresponding to the base of the detection signal is the lower limit. Can be set.
Note that the temperature range ΔTW and the temperature range ΔTM may be set to have the same width (for example, 10 ° C.) or different widths (for example, the temperature range ΔTW is 10 ° C. and the temperature range ΔTM is 7 ° C.). Further, the temperature range ΔTW and the temperature range ΔTM may be set such that the respective ranges from the melting temperature Tm to plus X ° C. and minus X ° C. (X ° C. is within 15 ° C., preferably within 10 ° C., for example).

  Next, for each of the temperature range ΔTW and the temperature range ΔTM, an area surrounded by a straight line passing through a point corresponding to the lower limit and a point corresponding to the upper limit of the temperature range of the differential melting curve and the differential melting curve (FIG. 1 ( (B) The hatched portion) is obtained. As an example of how to obtain the area, it can be specifically obtained as follows. The differential value of the detection signal at the temperature T is set to f (T), and the base value at the temperature T is set to B (T) by the following equation (1).

Area S = {f (Ts + 1) −B (Ts + 1)} + {f (Ts + 2) −B (Ts + 2)} + {f (Te−1) −B (Te−1)} (1)
However, Ts is a lower limit value in each temperature range, and Te is an upper limit value. Further, the base value B (T) at each temperature T is a value obtained by the following equation (2) and represents the background level included in the detection signal. By subtracting this base value from the differential value of the detection signal, the influence of the background included in the detection signal is removed.

B (T) = a × (T−Ts) + f (Ts) (2)
However, a = {f (Te) −f (Ts)} / (Te−Ts).

According to the above formulas (1) and (2), the area SW in the temperature range ΔTW and the area SM in the temperature range ΔTM are determined for the nucleic acid mixture, and the wild type codon 13 and the G13D mutant codon 13 are determined by area ratio. The presence or absence can be determined.
The area ratio can be obtained by the following equation (3).

Area ratio (%) = (SM / SW) × 100 (3)
For example, the presence or absence of the G13D mutant of the K-ras gene is determined by setting the area ratio cut-off value of the above formula (3) to 10%, and if it is 10% or more, the G13D mutant codon 13 You may determine that it exists (positive). The cut-off value may be set as appropriate according to the desired detection sensitivity and characteristics.

  When quantitatively measuring the abundance ratio of the wild-type codon 13 of the K-ras gene and the codon 13 of the G13D mutant in the sample, the melting curve and differential melting obtained using the actual sample The area ratio may be calculated from the curve, and the abundance ratio of the base sequence having the polymorphism contained in the actual sample may be determined based on a calibration curve prepared in advance.

  The calibration curve is created by, for example, preparing a plurality of nucleic acid mixtures in which the abundance ratios of two types of nucleic acids, wild type nucleic acid Wt and mutant type nucleic acid Mt, are different, and melting each of the plurality of nucleic acid mixtures. Get a curve. Subsequently, for each nucleic acid mixture, the area SW in the temperature range ΔTW and the area SM in the temperature range ΔTM are determined for each nucleic acid mixture according to the above formulas (1) and (2), and the relationship between the area ratio and the abundance ratio of each nucleic acid mixture is expressed. Create a calibration curve. For example, a calibration curve can be obtained by taking the abundance ratio (ratio of nucleic acid Mt to the total amount of nucleic acid mixture) on the horizontal axis and the area ratio (SM / SW) on the vertical axis. The calibration curve may be created by taking the abundance ratio (ratio of nucleic acid Mt to the total amount of nucleic acid mixture) on the horizontal axis and the area ratio (SM / SW) on the vertical axis. The area ratio may be determined by SW / SM.

Hereinafter, probes (polymorphism analysis probes) used in the polymorphism analysis method of the present invention will be described in detail.
The probe for polymorphism analysis is not particularly limited, and can be set by a conventionally known method. For example, the detection target sequence including the gene polymorphism site may be designed based on the sequence of the sense strand of the K-ras gene, or may be designed based on the sequence of the antisense strand. Moreover, it may be designed so as to bind to a region including a gene polymorphic site, so that the affinity with each of the wild type and the mutant type is different. Or you may design so that it may couple | bond with the area | region which does not contain a gene polymorphic site. A person skilled in the art can design a suitable probe by a conventionally known method according to the method of polymorphism analysis to be applied.

  For example, since the 224th base in SEQ ID NO: 6 is G, it is the codon 13 of the wild type K-ras gene, and A is the G13D mutant, so that the base corresponding to the 224th base in SEQ ID NO: 6 A probe (sense strand detection probe) complementary to either the detection target sequence in which G is G and the detection target sequence in which the base corresponding to the 224th position of SEQ ID NO: 6 is A, and the sequence of the antisense strand A probe complementary to (antisense strand detection probe) can be used.

  Although the specific example of a probe is shown in Table 1, it is not limited to this. The probes in Table 1 are probes for detecting the antisense strand of the G13D mutant K-ras gene. In the base sequences shown as SEQ ID NOs: 8 to 13, A shown in capital letters is a base complementary to the polymorphic site of codon 13 of the G13D mutant K-ras gene.

  The probe for polymorphism analysis of codon 13 of the K-ras gene shown in Table 1 is preferably labeled with a labeling substance such as a fluorescent dye as described above. Further, it is preferable to add a phosphate group to the 3 'end. As a specific example of the probe for polymorphism analysis, a complementary strand of the oligonucleotide shown in Table 1 may be used.

<Drug efficacy determination method>
The drug efficacy determination method according to one embodiment of the present invention (hereinafter simply referred to as “the drug efficacy determination method of the present invention”) is to analyze the polymorphism of codon 13 of the K-ras gene by the polymorphism analysis method of the present invention. And determining the efficacy of the drug based on the analysis result in the above analysis.

As described above, according to the polymorphism analysis method of the present invention, using the nucleic acid in the sample as a template, the region containing the gene polymorphic site of codon 13 of the K-ras gene is simultaneously highly efficient and wild type in the same reaction solution. Alternatively, it is possible to specifically amplify each of the G13D mutants and analyze the polymorphism of codon 13 of the K-ras gene. It is known that when the codon 13 of the K-ras gene is a G13D mutant type, the anti-EGFR antibody drug has a lower efficacy than that of the wild type. Therefore, according to the drug efficacy determination method of the present invention, the drug efficacy can be determined based on the polymorphism analysis result of codon 13 of the K-ras gene.
The obtained determination result can be used for prediction of the effect of a drug, and can also be used for determination of a treatment policy including change of dosage, switching to another drug, and the like.
For example, when the codon 13 of the K-ras gene is a G13D mutant, it is predicted that the anti-EGFR antibody drug has a low drug effect, and therefore switching to another drug can be performed. Predicting the effect of a drug, changing dosage, switching to another drug, etc., combined with determination of the presence or absence of other mutations in the K-ras gene and genotypes of other related genes You may go.
In addition, the drug is not limited to an anti-EGFR antibody drug, and any drug may be used as long as the drug effect is different depending on whether the codon 13 of the K-ras gene is a wild type or a G13D mutant.

<K-ras gene amplification kit>
A kit for amplifying a K-ras gene according to an embodiment of the present invention (hereinafter, simply referred to as “kit of the present invention”) includes the forward primer set of the present invention, and is obtained by a nucleic acid amplification method using K-ras. Used to amplify the region of interest containing codon 13 of the gene. By including the forward primer set of the present invention in the kit of the present invention, the region containing the gene polymorphic site of codon 13 of the K-ras gene is efficiently amplified with high specificity to each of the wild type and G13D mutant types. can do.

The kit of the present invention preferably contains a reverse primer used in a gene amplification method using the forward primer set of the present invention.
In order to detect the amplification product obtained by the gene amplification method using the forward primer set of the present invention, the kit of the present invention preferably further includes each probe capable of hybridizing to the amplification product. This probe is preferably a fluorescently labeled probe as described above. About a reverse primer and a probe, the matter regarding the reverse primer and probe mentioned above can be applied as it is.

In the forward primer set (1), (2) or (3) included in the kit of the present invention, the two forward primers included in each forward primer set may be separately accommodated or may be a mixture. Good. In addition, the forward primer set according to an embodiment of the present invention and the reverse primer or the probe may be separately accommodated or may be a mixture.
Note that “separately accommodated” is not limited as long as each reagent is divided so that a non-contact state can be maintained, and is not necessarily accommodated in an individual container that can be handled independently.

  For example, the reagents may be contained in a dissolved state in a buffer solution, or may be contained as a lyophilized product. As a container for storing the reagents, for example, a glass or plastic vial can be used.

  In addition to the above, the kit of the present invention may further contain various reagents necessary for the K-ras gene amplification method of the present invention or the polymorphism analysis method of the present invention. The kit of the present invention also includes instructions for use of the gene amplification method of the present invention or the polymorphism analysis method of the present invention, and various reagents included in or additionally contained in the kit of the present invention. It may further include written instructions for use.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

  Each primer shown in Table 2 was evaluated as a forward primer for specifically amplifying a nucleic acid in which codon 13 of the K-ras gene is a wild type or a mutant type. All of these primers were prepared according to a conventional method.

In Table 2, “wild-type specific amplification primer” is a forward primer for specifically amplifying the K-ras gene whose codon 13 is wild-type, and “G13D mutant-specific amplification primer” , A forward primer for specifically amplifying the K-ras gene in which codon 13 is a G13D mutant.
All combinations of 4 types of wild type specific amplification primers and 3 types of G13D mutation type specific amplification primers were evaluated.

  In the following examples, PCR and Tm analysis were performed using a reaction solution shown in Table 3 using a gene analyzer (trade name i-densy IS-5320, manufactured by ARKRAY).

The probe used was 5FL-KrasG13Dmt-F6 (SEQ ID NO: 12). Table 4 shows details of the probe 5FL-KrasG13Dmt-F6. In the sequence shown in Table 4, (FL) indicates that 5'-terminal cytosine (C) is labeled with BODIPY FL (registered trademark, manufactured by Molecular Probes, detection wavelength: 515 nm to 555 nm), which is a fluorescent dye. P indicates that a phosphate group is added to the 3 ′ end. The fluorescent labeling at the 5 ′ end and the addition of a phosphate group to the 3 ′ end were carried out according to conventional methods.
Moreover, in the base sequence shown in Table 4, the capital letter base indicates that it is a base corresponding to the G13D gene polymorphic site of codon 13 of the K-ras gene.

  As a template for nucleic acid amplification, (1) a template corresponding to a K-ras gene having a wild-type codon 13 (wild-type template) (trade name Human Genomic DNA, Roche Diagnostics GmbH), (2) wild-type Primers were evaluated using two types of templates: a template in which G13D mutant plasmid DNA was added so that the copy number was 0.3% (0.3% mutant template). Plasmid DNA used for the 0.3% mutant template is Genbank Accession No. PUC57 vector (GenScript) containing the sequences of NG — 007524, 10380-10699. The number of copies of the template used was 10,000 copies per measurement for both the wild type template and the 0.3% mutant template.

  The reverse primer described in Table 5 was used.

Using the prepared reaction solution, PCR and Tm analysis were performed by a fully automatic SNPs inspection device (trade name i-densy IS-5320, manufactured by Arkray).
The PCR was repeated at 50 ° C. for 50 seconds after treating at 95 ° C. for 60 seconds, followed by 95 ° C. for 1 second and 58 ° C. for 15 seconds.
Tm analysis was performed at 95 ° C. for 1 second and at 40 ° C. for 60 seconds, followed by increasing the temperature from 40 ° C. to 95 ° C. at a rate of temperature increase of 1 ° C./3 seconds, and the change in fluorescence intensity over time during that time. Was measured. In addition, the excitation wavelength of the fluorescent dye BODIPY FL in Tm analysis is 420 nm to 485 nm, and the detection wavelength is 520 nm to 555 nm.

Area ratio (%) = (Area of mutant type specific peak / Area of wild type specific peak) × 100
In the formula, “wild type specific peak” is a peak specific to the K-ras gene in which codon 13 is a wild type in a differential melting curve of Tm value analysis, and “mutant type specific peak” is codon 13 Is a peak specific to the K-ras gene which is a G13D mutant. The peak area was calculated as described above. In the above-described method, the temperature range ΔTW and the temperature range ΔTM are both 6 ° C.
When the area ratio (%) obtained by the above formula is 10% or more, the G13D mutant K-ras gene is present (positive), and when it is less than 10%, the G13D mutant K-ras gene is present. It was determined that it was not present (negative).

Table 6 shows the determination results for each of the forward primer sets for the wild type template and the 0.3% mutant template. “False positive” in the comprehensive determination means that the G13D mutant K-ras gene is determined to be positive when a wild-type template that does not include a K-ras gene having a G13D mutant is used. “False negative” was correctly determined that the G13D mutant K-ras gene was negative when the wild type template was used, but the G13D mutant K-ras when the 0.3% mutant template was used. It means that the presence of the gene could not be detected and the result was judged negative. The “correct answer” is that the G13D mutant K-ras gene is correctly determined to be negative when the wild type template is used, and the G13D mutant K-ras is used when the 0.3% mutant template is used. It means that the gene was correctly determined as positive.
A differential melting curve obtained by Tm value analysis using a wild type template is shown in FIG. 2, and a differential melting curve obtained by Tm value analysis using a 0.3% mutant template is shown in FIG. .

(1) Wild type specific primer WT-F6 and mutant type specific primer Mt-F3, (2) Wild type specific primer WT-F10 and mutant type specific primer Mt-F5 or (3) Wild type specific primer The forward primer set of WT-F10 and the mutant-specific primer Mt-F8 can detect even a mutant template that does not show false positives and is contained in only 0.3% of the wild-type template, and is correct. The result was obtained.
Moreover, the fact that codon 13 containing 0.3% copy number with respect to the total copy number of K-ras gene can detect K-ras gene having a G13D variant without false positives. It shows that the K-ras gene in which the codon 13 derived from the tumor present therein is a G13D mutant can be detected to the extent that it can be used clinically.

Claims (13)

The forward primer set for amplifying a K-ras gene for amplifying a region containing the codon 13 of the K-ras gene by the nucleic acid amplification method, which is a forward primer set of the following (1), (2) or (3):
(1) a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 1, a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 3,
Forward primer set, including
(2) a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 2, a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 4,
Forward primer set, including
(3) a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 2, a forward primer that is an oligonucleotide consisting of the base sequence of SEQ ID NO: 5,
Forward primer set.   A K-ras gene amplification method using the forward primer set for K-ras gene amplification according to claim 1.   The method according to claim 2, comprising amplifying a region containing codon 13 of the K-ras gene using the nucleic acid in the sample as a template and the forward primer set for amplifying the K-ras gene in one reaction solution. -A ras gene amplification method. Amplifying a region containing codon 13 of the K-ras gene by the K-ras gene amplification method according to claim 2 or 3,
Forming a hybrid between a single-stranded nucleic acid obtained from the amplification product of the amplification and a probe hybridizing to the single-stranded nucleic acid obtained from the amplification product;
Changing the temperature of the reaction solution containing the hybrid, and measuring the signal variation based on the dissociation state of the hybrid;
Analyzing a polymorphism of codon 13 of the K-ras gene based on the variation of the signal.   The polymorphism analysis method according to claim 4, wherein the amplification and the formation of the hybrid proceed simultaneously.   The polymorphism analysis method according to claim 4 or 5, wherein the probe is a probe that hybridizes to a region containing codon 13 of the K-ras gene.   The polymorphism analysis method according to any one of claims 4 to 6, wherein the probe is a fluorescently labeled probe. Analyzing the polymorphism of codon 13 of the K-ras gene by the polymorphism analysis method according to any one of claims 4 to 7;
Determining the efficacy of the drug based on the analysis result obtained by the analysis.   The method according to claim 8, wherein the drug is an anti-epidermal growth factor receptor antibody drug.   A kit for amplifying a K-ras gene for amplifying a region containing the codon 13 of the K-ras gene, comprising the forward primer set for amplifying the K-ras gene according to claim 1.   The kit for amplifying a K-ras gene according to claim 10, comprising one or more reverse primers for amplifying a region containing codon 13 of the K-ras gene.   The kit for amplifying a K-ras gene according to claim 10 or 11, comprising a probe that hybridizes to a region containing codon 13 of the K-ras gene.   The kit for K-ras gene amplification according to claim 12, wherein the probe is a fluorescently labeled probe.