JP2014187904A - Method for detecting nucleotide polymorphism from blood - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting nucleotide polymorphism from blood directly and rapidly based on melting curve analysis using a DNA detecting agent without purification (pretreatment) of the blood.SOLUTION: By using a family B DNA polymerase with a DNA detecting agent and two or more allele-specific primers in a reaction solution, mutation or nucleotide polymorphism can be detected by adding blood to the reaction solution without purification (pretreatment) of the blood.

Description

The present invention relates to the field of nucleic acid amplification. More specifically, the present invention relates to a method for detecting nucleic acid sequence mutation or polymorphism using melting curve analysis. The present invention is useful not only for research but also for genetic disease diagnosis, nucleotide polymorphism analysis and the like as clinical diagnosis.

  In the present invention, the nucleic acid sequence mutation and nucleotide polymorphism refer to having a nucleotide sequence different from that of the wild type. Generally, a frequency of less than 1% is sometimes called a mutation, and a frequency of 1% or more is called a polymorphism, but here it is not intended to be used strictly. This nucleotide polymorphism is known to affect disease susceptibility, drug metabolism, and the like, and is used as a genetic marker for diseases, a drug effect, and a marker for predicting side effects. Therefore, elucidation of these nucleotide polymorphisms and mutations is clinically important, and various detection methods have been developed.

  Conventional nucleic acid sequence analysis techniques include, for example, nucleic acid sequencing (sequencing), TaqMan probe (Genome Res. Vol. 6, 986 (1996)), restriction enzyme cleavage length polymorphism (RFLP) ( J. Clin. Invest. 72, pp. 1262 (1983)), allele-specific primer (ASP) method (WO 01/042498), allele-specific oligo probe (ASO) method (Nature 324, 163) (1986)), Pyrosequencing method (Analytical Biochemistry Vol. 224, p. 367 (1997)), Invader method (Nature Biotech. Vol. 17, p. 292 (1999), Quenching probe / Primer (Primer) ) Method (Japanese Patent No. 3437816) is known.

  In order to simplify the detection, methods capable of detecting a mutation polymorphism with one tube have been developed, such as TaqMan probe method, ASO method, and QP method (Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3). . However, most of these require fluorescently modified oligonucleotides.

On the other hand, as a method that does not require a fluorescently modified oligonucleotide, Germer et al. Added a non-complementary sequence (26 base pair GC tail) to one allele-specific primer, thereby changing the Tm value of the amplified product. A method for generating and detecting polymorphism has been reported (Non-patent Document 3). In addition, in order to balance the peak ratios of the amplification products amplified by each allele-specific primer in the melting curve analysis, Germer et al. Have different lengths of non-complementary sequences, GC, for each allele-specific primer. A method for adding a tail has been invented (Patent Document 2, Non-Patent Document 4). In these methods, detection is possible by melting curve analysis using an intercalator without using fluorescently modified oligonucleotides.
However, although this invention is being simplified, it is necessary to perform detection using genomic DNA that has been subjected to pretreatment such as purification before amplification. For this reason, problems remain in the ease and speed of operation.

Japanese Patent No. 3437816 Japanese Patent No. 4351217

Genome Research, Vol. 6, 986-994 (1996) Nature, Vol. 324, pp. 163-166 (1986) Genome Research, Vol. 9, pp. 72-78 (1999) BioTechniques, 39, 885-893 (2005)

  Nucleotide polymorphism detection is clinically important as a genetic marker for disease, a marker for predicting the effects and side effects of administered drugs. Therefore, in these industrial applications where processing of a large number of samples is required, it is strongly desired that the reaction time is shortened, the cost is low, and that it can be used in general-purpose equipment. For this reason, the problem to be solved by the present invention is that nucleotide polymorphisms can be analyzed by melting curve analysis using a DNA detection agent such as an intercalator more directly directly from blood without performing blood purification (pretreatment). It is to provide a method for performing detection.

In view of the above circumstances, the present inventors have conducted a blood purification treatment (pretreatment) by using a DNA polymerase belonging to Family B in addition to a DNA detection agent and an allele-specific primer as a result of intensive studies. However, the inventors have found that mutations or nucleotide polymorphisms can be detected simply by adding blood directly to the reaction solution, and the present invention has been completed.
Conventional detection using a DNA polymerase belonging to family A typified by Taq DNA polymerase requires blood pretreatment, and it is difficult to detect nucleotide polymorphisms simply by adding blood as it is. In addition, when diluted blood is used, it is accompanied by a decrease in the amount of template, making it difficult to detect nucleotide polymorphisms. On the other hand, in the present invention, by newly using a DNA polymerase belonging to Family B, mutation or nucleotide polymorphism can be detected directly from blood without diluting the blood.

That is, the present invention has the following configuration.
[1] A method for detecting a mutation or nucleotide polymorphism of DNA contained in a sample, comprising the following steps (1) to (4):
(1) Step of preparing a composition containing (c) from (a) below (a) DNA polymerase belonging to family B (b) DNA detection agent (c) Allele specific primer (2) Genomic extraction from blood sample Amplification was performed in steps (4) and (3) in which the sample was added to the composition described in (1) without performing step (3) and the amplification reaction was performed in the reaction solution described in (2). A step of detecting a DNA mutation or nucleotide polymorphism [2] The method according to [1], wherein the DNA detection agent is an intercalator.
[3] The method according to [1] or [2], wherein the blood is 0.01 to 5% by volume of the total volume of the PCR reaction mixture.
[4] The method according to [1] or [2], wherein the blood is 1 to 5% by volume of the total volume of the PCR reaction mixture.
[5] The method according to any one of [1] to [4], comprising two or more types of allele-specific primers.
[6] The method according to [1]-[5], wherein two or more types of allele-specific primers are designed to give amplification products having different Tm values.
[7] The method according to any one of [1] to [6], wherein at least one of the two or more allele-specific primers is a primer to which a GC tail is added.
[8] The method according to any one of [1] to [7], wherein two or more types of allele-specific primers are primers to which GC tails having different lengths are added.
[9] The method according to any one of [1] to [8], wherein mutation or nucleotide polymorphism is detected based on a difference in Tm values of amplification products given by two or more allele-specific primers.
[10] The DNA polymerase belonging to Family B is a DNA polymerase selected from the group consisting of KOD, Pfu, Pfx, Vent, DeepVent, Tgo, Pwo, PrimeStar, and MightyAmp, according to any one of [1] to [9] the method of.
[11] The method according to any one of [1] to [10], wherein the DNA polymerase belonging to Family B is derived from Thermococcus kodakaraensis (KOD).
[12] A kit for amplifying DNA contained in a sample without detecting the genome from the blood sample and detecting the mutation or nucleotide polymorphism thereof, comprising the following (a) to (c) A kit containing things.
(A) DNA polymerase belonging to family B (b) DNA detection agent (c) allele-specific primer

  According to the present invention, mutation or nucleotide polymorphism can be detected by melting curve analysis using a DNA detection agent without blood purification treatment (pretreatment). As a result, mutations or nucleotide polymorphisms can be detected rapidly in a shorter time than in the past. The present invention can be widely used not only in research fields but also in diagnostic applications. In addition, since detection can be performed in a short time, it can be expected to be used in many industrial applications that require processing of a large number of samples.

In Example 1, it is a figure which shows the result of having detected ALDH2 SNP from the blood from which an addition ratio differs. In Example 2, it is a figure which shows the result of having detected ALDH2 SNP directly from the blood at the time of designing a GC tail and a primer from which amplification length differs.

  Hereinafter, the present invention will be described in detail. The method for directly detecting a nucleotide polymorphism from blood using a DNA detection agent in the present invention is characterized by using a DNA polymerase belonging to Family B, a DNA detection agent, and an allele-specific primer.

  KOD, Pfu, Pfx, Vent, DeepVent, Tgo, Pwo, and the like are listed as DNA polymerases belonging to the above-mentioned family B. Currently, KOD DNA polymerase (TOYOBO), PrimeSTAR (registered trademark) DNA polymerase, and MightyAmp (registered) (Trademark) DNA polymerase etc. can be purchased. The enzyme used in the present invention may be any DNA polymerase belonging to Family B, and is not particularly limited to the above-mentioned DNA polymerase. Among them, KOD DNA polymerase is particularly preferable.

The DNA polymerase used in the present invention may be a mutant having an amino acid sequence in which one or several amino acids are substituted, deleted, added or inserted in the amino acid sequence, and is not particularly limited. The mutant may be modified by introducing a mutation into a specific site by a known technical method.

The DNA detection agent used in the present invention is not particularly limited, and examples thereof include a dye that binds to a phosphate group on the DNA surface and a dye that intercalates between bases (intercalator), but it is preferable to use an intercalator.
An intercalator refers to any molecule that binds to a nucleic acid in a reversible, non-covalent manner by insertion (intercalation) into double-stranded DNA, thereby indicating the presence and amount of the nucleic acid. . In general, an intercalator is a dye that emits fluorescence when inserted into double-stranded DNA.

  A number of intercalators are known in the art. For example, Ethyl bromide, cyanine dyes (eg, TOTO, YOYO, BOBO and POPO), SYBR Green I, SYBR Green ER, SYBR Green Gold, SYBR DX, PicoGen, LC Gene, EvaG Acidine orange, 7-amino-actinomycin D, CyQUANT GR, SYTO-9, SYTO-10, SYTO-13, SYTO-14, SYTO-82, FUN-1, and the like are not particularly limited. .

In the present invention, it is preferable to use two or more types of allele-specific primers.
Two or more kinds of allele-specific primers in the present invention are used for detection of mutation or nucleotide polymorphism by melting curve analysis using an intercalator. Specifically, it is a method for detecting a base polymorphism contained in a nucleic acid sample based on whether or not DNA containing a specific base polymorphic site is amplified, and a blood sample containing DNA containing a specific base polymorphic site In addition, a common primer (corresponding to the forward primer) and two or more nucleotide polymorphism detection allele-specific primers (corresponding to the reverse primer) are simultaneously used for amplification reaction, and polymorphism is detected by melting curve analysis. It is a method to do.

Two or more types of allele-specific primers that detect nucleotide polymorphisms are amplified between the common forward primers using the target nucleic acid as a template only when a complementary sequence is found in the target template. In other words, mix allele-specific primer complementary to the first nucleotide polymorphism, allele-specific primer complementary to the second nucleotide polymorphism, common primer, only the first nucleotide polymorphism When PCR amplification is performed using the homozygote contained as a template, only amplification with an allele-specific primer complementary to the first nucleotide polymorphism is observed. Similarly, when a homozygote containing only the second nucleotide polymorphism is used as a template, only amplification with an allele-specific primer complementary to the second nucleotide polymorphism is observed. In addition, when a heterozygote containing both the first nucleotide polymorphism and the second nucleotide polymorphism is used as a template, allele-specific primers that are complementary to each other, that is, allele specific complementary to the first nucleotide polymorphism. Both amplification with a specific primer and amplification with an allele-specific primer complementary to the second nucleotide polymorphism are observed. That is, by using an allele-specific primer, nucleotide polymorphism is identified and nucleotide polymorphism-specific amplification is enabled.

  In addition, two or more types of allele-specific primers in the present invention are characterized by having different Tm values. Thereby, a difference arises in Tm value of an amplification product, and when melting curve analysis using an intercalator is performed, a peak will be separated. That is, the amplification product amplified by the allele-specific primer can be identified and detected.

  That is, the two or more types of allele-specific primers in the present invention are such that the polymorphism is identified and amplified as an allele-specific primer, and the Tm value of the amplification product amplified by each allele-specific primer is different. Thus, when the melting curve analysis is performed, the peak is divided by the difference in Tm value, and both of the side surfaces for identifying and detecting the base polymorphism are included.

  As the length of the primer in the present invention, the sequence complementary to the target nucleic acid is preferably 13 to 35 bases, more preferably 16 bases or more, and preferably 30 bases or less. On the other hand, in order to cause a difference in Tm value, it is desirable to add 2 to 40 bases of a sequence that is not completely complementary to DNA at the 5 ′ end of the allele-specific primer, more preferably 30 bases or less, even more preferably. Is 20 bases or less, more preferably 15 bases or less. A sequence that is not completely complementary to DNA may be added to only one type of allele-specific primer, so that a difference may be caused in the Tm value of the amplification product. However, in order to balance the amplification efficiency, it is more preferable. It is preferable to add sequences having different Tm values and not completely complementary to DNA to a plurality of allele-specific primers.

  The sequence that is not completely complementary to the DNA to be added is not particularly limited, but in order to cause a difference in the Tm value with a smaller number of bases, a guanine (G) or cytosine (C) base is added. It is desirable to include a GC tail. The GC tail added to the 5 ′ end of this primer preferably has a total amount of guanine (G) and cytosine (C) bases of 60% or more, more preferably 70% or more, still more preferably 80% or more, Particularly preferably, it is 90% or more. The GC tail may be added to only one type of allele-specific primer, but more preferably, while maintaining the balance of amplification efficiency, the difference in the Tm values of the amplification products amplified with a plurality of allele-specific primers. In order to generate it, it is desirable to add GC tails of different lengths to multiple allele-specific primers. The difference in the length of the GC tail is preferably 5 bases or more, more preferably 10 bases or more and 15 bases or less.

  Furthermore, the amplified chain length is preferably 300 bp or less, more preferably 200 bp or less, and particularly preferably 100 bp or less in order to cause a difference in the Tm value with a smaller number of bases.

  The feature of two or more types of allele-specific primers in the present invention is that a difference is caused in the Tm value of the amplification product amplified by each allele-specific primer. The Tm value of each amplification product is determined by each real-time PCR apparatus and analysis software. In addition, the melting temperature of the amplification product may be determined using a commercially available primer design program or an online program provided by a public institution such as the Oligo Calculator Basic method (http://www.basic.northwestern.edu/biotools/oligocalc.html). It can be easily estimated by using #helpermo). That is, in the allele-specific primer and the common primer in the present invention, the difference in the Tm value of the amplification product amplified by each allele-specific primer is 1 ° C or higher, preferably 3 ° C or higher, more preferably 5 ° C or higher. What is necessary is just to design.

In the allele-specific primer in the present invention, it is preferable that the third base from the 3 ′ end corresponds to each nucleotide expected at the nucleotide polymorphism site, more preferably the second base from the 3 ′ end. Is preferably a nucleotide polymorphism site.
Furthermore, it is preferable that at least one base from the 3 'end to the 5' end of the primer is substituted with a base (mismatch base) that is not complementary to the base of the strand to be hybridized. Is designed to have non-complementary bases 3 to 7 from the 3 'end. Further, it is particularly preferable that the non-complementary base is designed to have the third position from the 3 ′ end. The non-complementary bases in the primer can also be different bases in each primer.

  The greatest feature of the present invention is to detect mutations and nucleotide polymorphisms directly without performing genome extraction (pretreatment) from blood. The addition ratio of this blood in the PCR reaction solution is 0.01% by volume or more, preferably 0.1% by volume or more, more preferably 1% by volume or more with respect to the PCR reaction solution. Moreover, it is preferable that the upper limit of the said addition ratio is 5 volume% or less. The blood may be added directly to the PCR reaction solution, but may be diluted as appropriate and added to the PCR reaction solution. In addition, the form of blood added to PCR is not particularly limited, and for example, collected blood may be used as it is, or cryopreserved blood can be frozen and used. Further, for example, it may be any of hemolyzed blood, unhemolyzed blood, anticoagulated blood, blood containing a coagulated fraction, and the like.

Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1: Detection of ALDH2 SNP directly from blood (blood addition amount)
In this example, a sequence part known as an aldehyde dehydrogenase gene (ALDH2, 487 G → A) of a human genome sequence was selected as a specific gene for detecting a base polymorphism.
This example is an example in which ALDH2 SNP is detected by variously adding the blood sample in the PCR reaction solution. Amplification was performed using the common primer # 1 represented by SEQ ID NO: 1 and the allele-specific primers represented by SEQ ID NOs: 2 and 3 as primers.
G-specific primer # 1 (SEQ ID NO: 2) is an oligonucleotide for detecting [G], has a nucleotide sequence complementary to [G] second from the 3 ′ end, and has an artificial mismatch at the third. The A-specific primer # 1 (SEQ ID NO: 3) is an oligonucleotide for detecting [A], the nucleotide sequence complementary to [A] second from the 3 ′ end, and the third artificial mismatch It was supposed to have.
In addition, a 14-base GC tail is added to the G-specific primer # 1 (SEQ ID NO: 2), and an A-specific primer so that the Tm value between the amplification product by the G-specific primer and the amplification product by the A-specific primer is different. A 6-base GC tail was added to # 1 (SEQ ID NO: 3). Further, the Tm value of the amplification product is predicted using the basic method of the Tm value calculation program (http://www.basic.northwestern.edu/biotools/oligocalc.html#helpermomo) so that the difference in Tm value occurs. The position of common primer # 1 (SEQ ID NO: 1) was determined.

  In this example, KOD DNA polymerase (TOYOBO) was used as a DNA polymerase belonging to family B, and the PCR buffer was KOD-Plus-Ver. The one attached to 2 (TOYOBO) was used. In addition, as the intercalator, 1 / 30,000-fold diluted SYBR (registered trademark) Green I was used to prepare a PCR reaction solution. Then, it added to the said PCR reaction composition so that the addition ratio of the blood might be 5%, 2%, 1%, 0.5%, 0.1% of a PCR reaction liquid. The PCR reaction solution was subjected to amplification by PCR reaction and detection by melting curve analysis under the following conditions shown in Table 3 using a real-time PCR apparatus (Applied Biosystems 7500 Fast real-time PCR system).

In addition, the significance of using a DNA polymerase belonging to Family B was compared with a reagent using Taq DNA polymerase, which is a DNA polymerase belonging to Family A. Specifically, THUNDERBIRD SYBR qPCR Mix (TOYOBO), which is a SYBR Green I detection system real-time PCR reagent using Taq DNA polymerase, was used, and the same primers and templates as described above were used. PCR reaction conditions are as shown in Table 4 and Table 5 below.

  These results are shown in FIG. FIG. 1 shows the results of PCR and melting curve analysis at each blood addition amount. In the conventional detection using a DNA polymerase belonging to Family A, nucleotide polymorphisms could not be detected without genome purification (pretreatment) from blood. On the other hand, by using a DNA polymerase belonging to Family B in the present invention, it is possible to detect each genotype simply by adding blood to the PCR reaction solution so as to be 0.1% to 5% by volume. It was. That is, it was confirmed that the present invention can detect a nucleotide polymorphism directly from blood using an intercalator in the above primer designing method by using a DNA polymerase belonging to Family B.

Example 2: Detection of ALDH2 SNP directly from blood (when GC tail is added to one side and the effect of amplification length)
This example is different from Example 1 by selecting a sequence part known as an aldehyde dehydrogenase gene (Aldehyde dehydrogenase2: ALDH2, 487 G → A) of a human genome sequence as a specific gene for detecting a nucleotide polymorphism. This is an example in which ALDH2 SNP was detected with a primer set.
First, in the first primer set (Table 6), a GC tail is added only to one allele-specific primer. Common primer # 1 (SEQ ID NO: 1) and G-specific primer # 1 (SEQ ID NO: 2) were the same as in Example 1. Only the A-specific primer # 2 (SEQ ID NO: 4) was an allele with no GC tail added. Specific primers were used. This A-specific primer # 2 (SEQ ID NO: 4) is an oligonucleotide for detecting [A], which has a nucleotide sequence complementary to [A] second from the 3 ′ end, and an artificial mismatch third. It was supposed to have.

Next, the second primer set (Table 7) is a case where the amplification length is designed to be different. For the allele-specific primer, G-specific primer # 1 (SEQ ID NO: 2) and A-specific primer # 2 (SEQ ID NO: 4) were used. On the other hand, the common primer # 2 (SEQ ID NO: 5) was designed so as to increase the amplification length by changing the position of annealing with the template nucleic acid. This common primer # 2 (SEQ ID NO: 5) predicts the Tm value of the amplification product using the basic method of the Tm value calculation program (http://www.basic.northwestern.edu/biotools/oligocalc.html#helpermo) The difference in Tm value between the G-specific amplification product and the A-specific amplification product was set to 3 ° C.

In this example, as in Example 1, 1 / 30,000-fold diluted SYBR (registered trademark) Green I was used as an intercalator, and KOD DNA polymerase (TOYOBO) was used as a DNA polymerase belonging to Family B, in real time. Using a PCR device (Applied Biosystems 7500 Fast real-time PCR system), amplification by PCR reaction and detection by melting curve analysis were performed under the PCR conditions shown in Tables 2 and 3. In this example, blood was added to the PCR reaction composition so that the ratio of blood addition was 0.5% with respect to the PCR reaction solution.
These results are shown in FIG. FIG. 2 is a diagram showing the results of detecting ALDH2 SNP directly from blood when a GC tail and primer designs with different amplification lengths are designed. Whether the GC tail was added to only one of the allele-specific primers or the amplification length was increased, each genotype could be detected by adding blood directly. In other words, regardless of the GC tail addition method and the amplification length, the primer design can be made so that the Tm value of the amplified product differs, so that the base polymorphism can be detected directly from blood using an intercalator. confirmed.

  According to the present invention, mutation or nucleotide polymorphism can be detected by melting curve analysis using a DNA detection agent without performing blood purification treatment (pretreatment). Therefore, mutation or nucleotide polymorphism can be detected in a shorter time and at a lower cost. The method for detecting mutations or nucleotide polymorphisms directly from blood can be widely used not only for research purposes but also for genetic diagnosis. In addition, since it can be detected in a short time, it can be expected to be used in many industrial applications that require processing of a large number of samples.

Claims (12)

A method for detecting a mutation or nucleotide polymorphism of DNA contained in a sample, comprising the following steps (1) to (4):
(1) Step of preparing a composition containing the following (a) to (c) (a) DNA polymerase belonging to family B (b) DNA detection agent (c) Allele specific primer (2) Genome extraction from blood sample Amplification was performed in steps (4) and (3) in which the sample was added to the composition described in (1) without performing step (3) and the amplification reaction was performed in the reaction solution described in (2). Step of detecting DNA mutation or nucleotide polymorphism   The method according to claim 1, wherein the DNA detection agent is an intercalator.   The method according to claim 1 or 2, wherein the blood is 0.01 to 5% by volume of the total volume of the PCR reaction mixture.   The method according to claim 1 or 2, wherein the blood is 1-5% by volume of the total volume of the PCR reaction mixture.   The method in any one of Claims 1-4 containing two or more types of allele specific primers.   The method according to claim 1-5, wherein two or more allele-specific primers are designed to give amplification products of different Tm values.   The method according to any one of claims 1 to 6, wherein at least one of the two or more types of allele-specific primers is a primer to which a GC tail is added. The method according to any one of claims 1 to 7, wherein the two or more types of allele-specific primers are primers to which GC tails having different lengths are added.   The method according to any one of claims 1 to 8, wherein a mutation or a nucleotide polymorphism is detected by a difference in Tm values of amplification products given by two or more kinds of allele-specific primers.   The method according to any one of claims 1 to 9, wherein the DNA polymerase belonging to Family B is a DNA polymerase selected from the group consisting of KOD, Pfu, Pfx, Vent, DeepVent, Tgo, Pwo, PrimeStar, and NightyAmp.   The method according to any one of claims 1 to 10, wherein the DNA polymerase belonging to Family B is derived from Thermococcus kodakaraensis (KOD). A kit for amplifying DNA contained in a sample without performing genome extraction from the blood sample and detecting the mutation or nucleotide polymorphism thereof, comprising a composition comprising the following (a) to (c) kit.
(A) DNA polymerase belonging to family B (b) DNA detection agent (c) allele-specific primer