WO2002024905A1

WO2002024905A1 - Method of judging gene mutation

Info

Publication number: WO2002024905A1
Application number: PCT/JP2001/007942
Authority: WO
Inventors: Nobuyuki Hamajima
Original assignee: Nobuyuki Hamajima
Priority date: 2000-09-19
Filing date: 2001-09-13
Publication date: 2002-03-28
Also published as: AU2001286218A1; JP2001137000A

Abstract

A method of economically and quickly judging a mutation in a nucleic acid on gene is provided. Namely, a method of judging the presence or absence of a specific gene mutation which comprises confirming the size of DNA fragments formed by a PCR reaction with the use of four primer DNAs including a primer DNA having a nucleic acid corresponding to the mutated nucleic acid at the 3'-end, a primer DNA having a nucleic acid corresponding to the normal nucleic acid at the 3'-end, and two primer DNAs corresponding respectively to these primer DNAs.

Description

Description Method for discriminating gene mutations Technical field

The present invention provides a method for easily discriminating the presence or absence of a gene mutation on a chromosome assumed in advance and the type on an allele.

Background art

Conventionally, it has been known that there are groups that exhibit a constitution that is susceptible to a certain disease, and groups that exhibit a constitution in which a certain degree of medicinal effect cannot be achieved. Due to recent advances in genomic analysis and rapid progress in human genome analysis, many of these phenomena are often replaced by single base substitutions in the base sequence of a specific gene. A great deal of knowledge has been accumulated that it is due to minor mutations (Single Nucleotide Polymorphisms, SNPs). At present, such SNPs are being vigorously analyzed. It has long been known that the onset of a disease is caused by a mutation in the nucleotide sequence of a gene.However, the progress of genomic analysis mentioned above has led to the development of mutations in genes that cause disease. New knowledge is also being rapidly increased. When considering genomic research from a clinical perspective, it is very important to quickly determine whether an individual has a mutation in a particular gene. It is expected that an effective prevention plan for the disease will be possible if it is possible to know in advance the diseases that an individual is susceptible to by this discrimination. In addition, if correlation data between those who have such a genetic mutation and a drug that is effective for that person is accumulated, it will be possible to develop more appropriate drugs or therapeutic methods for individuals who have such a mutation. There are significant benefits to clinical practice, such as being possible and leading to early detection of disease causes.

Several methods have been developed to determine the presence or absence of a mutation in a gene. PCR-RFLP combining the polymerase chain reaction (PCR) method and restriction enzyme cleavage (Erlich et al., Science, 255, 1643, pp. 1991) ) Is a typical example. Also, the DNA microarray method has been developed very recently (Brown et al., Nat. Gent. ^ 21, p. 33, 1999).

However, the PCR-RFLP method cannot be said to be a rapid method because the testing process involves a restriction enzyme treatment of 3 to 24 hours. Also, The DNA microarray method is extremely expensive to carry out, and is very disadvantageous in terms of processing a large number of samples. Therefore, there has been a demand for the development of a new method for discriminating gene mutations that can rapidly and inexpensively process a large amount of samples.

Means for solving the problem

The present inventors can utilize a large number of samples quickly and inexpensively by taking advantage of the PCR method as it is, and further utilizing the restrictions on primer DNA sequence design, which should be noted when performing the PCR method. A new method for identifying gene mutations (Confronting two-pair primers PCR) that can be processed in a single step has been developed.

That is, the present invention provides a primer DNA having a nucleic acid corresponding to a mutant nucleic acid at the 3 ′ end, a nucleic acid corresponding to a normal nucleic acid at 3 ′, a primer DNA having an end at a 3 ′ end, and two types thereof facing the same. This is a method for determining the presence or absence of a predetermined gene mutation by confirming the size of a DNA fragment generated by a PCR reaction using four types of primer DNAs containing the same primer DNA.

The present invention utilizes at least two sets of primer DNA sequences that are designed to have a desired base sequence in advance. The outline of the present invention is based on the target gene for which the presence or absence of a mutation is to be determined. Figure 1 shows an example of SNPs assuming that the base on allele X is X and the nucleic acid corresponding to base X on allele Y is Y.

First, two pairs of primers DNA are designed as four types of primers DNA. One set is a set for which allele X is to be amplified, and another set is a set for which allele Y is to be amplified. Among the set for allele X, one primer DNA (antisense primer 1R) is synthesized as the nucleic acid X 'complementary to the nucleic acid X, and the other end is synthesized as the nucleic acid X'. Synthesize primer 1F and combine them into one. On the other hand, for the allele Y, a sense primer DNA (primer 1F) whose 3 end is a nucleic acid Y is synthesized, and further downstream from the primer 2F. The antisense primer DNA (primer 2R), which is located opposite to the primers, is synthesized to form another combination.

If a PCR reaction is performed using only each set as a single primer DNA under the above conditions, the size of the specific DNA amplification product observed after each reaction is a base pair (size a ), B is a base pair (size b). Here, size a and size b need to be distinguished in molecular weight by a suitable method such as gel electrophoresis. However, this may be achieved by arbitrarily adjusting the position of the primer DNA on the target gene so as to give a desired size difference.

According to the present invention, a PCR reaction is carried out on a gene suspected of having a mutation at the same time using the two sets of primer DNAs described above, and the size of the PCR product is confirmed. The presence or absence of mutation and the type of mutation between alleles can be determined.

When the base in question on both alleles is type XX, the DNA between primer 1F and antisense primer 1R is amplified without any problem, and the size a node is confirmed. . On the other hand, the primer reaction does not proceed from the primer F that hybridized to the allele Y because the 3 ′ terminal base of the primer F is Y, and as a result, the primer F There is no amplification between the primer and the primer 2R. Primer 1: Since c-base pair (size c) DNA is amplified by PCR without any problem between F and primer 2R, an amplification band confirmed after PCR reaction The size of the code is size a and size c. In other words, when both the band of size a and the band of size c are specifically amplified by the PCR reaction according to the present invention, the bases that are problematic in the gene are both X alleles. Can be determined. The problematic base on both alleles is YY According to the same theory as above, the size of the band confirmed after the PCR reaction is the size b and size c nodes, which means that the size b and size c When the band is specifically amplified, it means that the base in question in the gene can be determined to be Y for both alleles. If, after the PCR reaction, three types of nodes, size a, size b, and size c, are observed, the base on the allele X becomes X based on the same principle as described above. Where c = a + b-(d-1) where the base on the allele Y can be determined to be a heterozygote of Y. However, d is the sum of the number of bases of both 1R and 2F primers.

Here, for allele X, nucleic acid X ′ is 3 and antisense primer 1R at the end is replaced with nucleic acid X3 and sense primer 2F at the end is replaced with antisense primer 1F. Combination of primer 2R and anti-primer having nucleic acid Y at the 3 'end instead of sense primer 2F having nucleic acid Y at the 3' end for allele Y The present invention can also be carried out by combining 1R and sense primer 1F and performing a PCR reaction in the coexistence of these. In this case, the size of the transcript is also size a, size b, and size c. When the size c is YY type, the size a and the size c are respectively confirmed. The nucleic acid corresponding to the mutant nucleic acid and the nucleic acid corresponding to the normal nucleic acid according to the present invention mean the mutant nucleic acid and the normal nucleic acid or the nucleic acid complementary thereto as in the above two examples.

In the PCR-CTPP method of the present invention, as described above, the PCR reaction and the confirmation of the molecular weight of the PCR product are completed in two steps, and the PCR product is treated with the restriction enzyme over several hours. Must be performed PCR-Compared to the RFLP method, the time required for discrimination can be significantly reduced. In addition, in the PCR-RFLP method, there is a restriction that a restriction enzyme site must be designed in advance so that a fragment having a molecular weight difference required for fractionation after restriction enzyme treatment is generated. Therefore, it may be difficult to design an appropriate primer DNA depending on the nucleotide sequence of the target gene, but the PCR-CTPP method of the present invention has no such restrictions at all. It also has an advantage in the flexibility of primer-DNA design. Furthermore, in the method of the present invention, if there is an artificial mistake such as a failure to add the primer 1F and the primer 2R to the reaction solution, a band of c-1 bp is not observed, and Testers can easily make mistakes You can get to know them. Furthermore, since the present invention does not require cleavage of a PCR product with a restriction enzyme, it is not affected by various artificial by-products due to the restriction enzyme treatment, and the determination can be performed reliably. Can be done.

The PCR-CTPPP method of the present invention has an advantage even compared to the DNA microarray method. In other words, no special equipment is required, and the cost difference when processing a large amount of specimens is significantly lower than that of the DNA microarray method.

The present invention is useful for discrimination of so-called polymorphism, but can also be used for discrimination of a causative gene or a cancer gene of a specific disease, and mutation is substitution, addition, or deletion. Any of the above is applicable.

Embodiment of the Invention

DNA containing the target gene to be discriminated can be subjected to a PCR reaction from blood or other tissues collected from a patient or a healthy person by a method commonly used by those skilled in the art in the field to which the present invention belongs. The present invention can be carried out using this as a sample as long as it is prepared as a DNA of the order.

The design of the primer DNA used in the method of the present invention is based on three terminal Besides the determination of the nucleic acid in step 1 and that the difference between the molecular weight (base pair number) of the amplified DNA product and the PCR product when using the primer DNA is appropriate. There are no separate restrictions. The size difference between the DNA product and the size of the PCR product can be appropriately determined depending on the sensitivity of the method for detecting the amplified product, but it is sufficient if there are tens to hundreds of base pairs in general. In the case of using a high-sensitivity detection method such as capillary-electrophoresis, the difference may be several base pairs. Such a primer DNA design can be easily performed by a general person skilled in the art to which the present invention belongs based on the nucleotide sequence of the gene to be identified and the position of the mutation. It is something. In addition, the preparation of the primer DNA itself can be easily performed using a so-called automatic DNA synthesizer or other general-purpose equipment or a synthesis method.

The PCR reaction in the present invention can also be performed under general reaction conditions that do not include special reagents or reaction steps, and commercially available PCR reactions can be used.

General-purpose equipment such as a CR reaction converter and a cycler can be used. In addition, the PCR reaction is performed using the primer D N

It is preferable to add all of A to the reaction system at the same time, and although the efficiency is reduced, as long as the primers coexist, each primer set It is also possible to carry out the PCR reaction step by step.

The PCR product can be detected by any method available to those skilled in the art, such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis. , High-performance liquid chromatography (HPLC) or gas chromatography, such as chromatography, mass spectrometry (GC-MS), etc. . Gel electrophoresis is preferred as a simpler method.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to those described in Examples.

Example

Example 1-Adrenoreceptor-2 (BAR-2) Polymorphism Detection of Adrenoceptor 2 (BAR-2), which has been reported to be involved in obesity and / or metabolic dysfunction Testing for polymorphism was performed. In this polymorphism, the nucleobase changes from C to G, and the 27th glutamine of the receptor protein is mutated to glutamic acid. A peripheral blood sample of 7 ml was prepared from 20 patients who gave written consent. Using a QIAampDNA Blood Mini Kit manufactured by Qiagen, chromosomal DNA was extracted from the buffy coat fraction and used as the chromosomal DNA in the following tests.

The primer DNA of SEQ ID NO: 1 was synthesized as the primer DNA for the allele of the normal nucleic acid C, and the primer DNA of SEQ ID NO: 2 was synthesized as the antisense primer DNA. In addition, the primer DNA of SEQ ID NO: 3 was synthesized as the sense primer DNA for the allele of the mutant nucleic acid G, and the primer DNA of SEQ ID NO: 4 was synthesized as the antisense primer: DNA. 30 ng to 100 ng of the chromosomal DNA prepared above was combined with 0.15 mM dNTP, 25 pmol of each primer DNA, and 0.5 unit of Evening Kara Shuzo T aq (R) enzyme, 1 5 m M of M g C l ₂ and including 2. 1 0 XPCR buffer 5 〃 1, and 2 5 1 of the reaction solution containing the 1 〃 1 of grayed Li Cerro Lumpur And a PCR reaction was performed. The reaction conditions were denaturation at 94 ° C for 5 minutes, followed by 30 cycles of 30 seconds at 94 ° C, 30 seconds at 59 ° C, and 30 seconds at 72 ° C. Finally, a final extension reaction is performed at 72 ° C for 5 minutes. PCR products were stained with 2% agarose gel and ethidium Visualized by. The result is shown in figure 2.

According to this method, a normal homozygous (CC type) in which two kinds of PCR products of 279 base pairs and 204 base pairs are observed, and 279 base pairs, 204 base pairs, 11 Heterotype (CG type) in which three types of PCR products of 0 base pairs are observed, and mutant homo type (GG type) in which two types of PCR products of 279 base pairs and 110 base pairs are observed ) Could be distinguished from each other.

• Example 2 Inspection for polymorphism in IL-1B (IL-IB) polymorphism Inspection for polymorphism where the 1st to 31st nucleic acid of IL-1B is changed from C to T Was. This polymorphic form of IL-1B is said to increase the risk of gastric cancer.

The primer DNA of SEQ ID NO: 5 was synthesized as the sense primer DNA for the allele of the normal nucleic acid C, and the primer DNA of SEQ ID NO: 6 was synthesized as the antisense primer DNA. In addition, a primer DNA of SEQ ID NO: 7 was synthesized as a primer DNA for an allele of a different nucleic acid T, and a primer DNA of SEQ ID NO: 8 was synthesized as an antisense primer DNA. From 30 ng of chromosomal DNA prepared in Example 1: LOO ng to 0.15 mM dNTP, 25 pmol of each primer DNA, 0.5 unit Evenings Kara Shuzo Ltd. T aq (TM) enzyme was added PCR reactions 1 2 containing 5 mM of M g C l _2. The reaction solution 2 5 ^ 1 comprising 1 0 XPCR buffer. The reaction conditions were denaturation at 94 ° C for 5 minutes, followed by 25 cycles of 60 seconds at 94 ° C, 60 seconds at 54 ° C, and 60 seconds at 72 ° C. Finally, a final extension reaction is performed at 72 ° C for 5 minutes. PCR products were visualized by 2% agarose gel and ethidium bromide staining. Figure 3 shows the results.

According to this method, a normal homozygous (CC type) in which two kinds of PCR products, 240 base pairs and 155 base pairs, are observed, 240 base pairs, 155 base pairs, and 125 base pairs Hetero-type (CT type) in which three types of PCR products of 2 base pairs are observed, and mutant homo-type (TT type) in which two types of PCR products of 240 base pairs and 122 base pairs are observed ) And could be distinguished from each other.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram schematically illustrating the principle and scheme of the method of the present invention.

FIG. 2 is an electrophoresis photograph of the PCR reaction product performed in Example 1. Lane M is a 100 base pair size marker, lane 2, 4, 6, 7, 9, 11, 11, 12, 15, 17, 20 are normal. Tero type (CC type), lanes 1, 5, 8, 13, 13, 14, 18 , 19 are heterotypes (CG type), and lanes 3, 10, and 16 are mutant homotypes (GG type).

FIG. 3 is an electrophoresis photograph of the PCR reaction product performed in Example 2. Lane M is a 100 base pair size marker, lanes 13, 15, and 18 are normal heterotype (CC type), lanes 1, 2, 4, 5, 7, 9, 9 and 1 0 and 14 are heterozygous (CT type), lanes 3, 6, 8, 11, 12, 16, 17, 19 and 20 are mutant homozygous (TT type) . .

Claims

The scope of the claims

1. A method for determining the presence or absence of a predetermined gene mutation by confirming the size of a DNA fragment generated by performing a PCR reaction in the presence of at least two sets of primer DNAs. Thus, one set of the primer DNA is composed of a primer DNA having a nucleic acid corresponding to the mutant nucleic acid at the 3 'end and a primer DNA facing the primer DNA, and the other set is composed of a normal nucleic acid at the 3' end. A method for determining the presence or absence of a gene mutation consisting of a primer having the corresponding nucleic acid and DNA facing the primer.

2. The method according to claim 1, wherein the opposing primers DNA of each set also constitute a set of opposing primers DNA.

3. The method according to claim 1 or claim 2, wherein the sizes of the DNA fragments generated in the PCR reaction have a discernable size difference from each other.