WO2001012849A1

WO2001012849A1 - Method of distinguishing nucleic acids and kits for nucleic acid test

Info

Publication number: WO2001012849A1
Application number: PCT/JP2000/005286
Authority: WO
Inventors: Akio Yamane
Original assignee: Wakunaga Pharmaceutical Co., Ltd.
Priority date: 1999-08-12
Filing date: 2000-08-07
Publication date: 2001-02-22
Also published as: JP2003174882A; AU6320400A

Abstract

A method of accurately and quickly detecting and quantitating a variation or polymorphism in a number of genes contained in a number of specimens by a convenient operation to thereby examine the presence/absence of a nucleic acid having a variation or polymorphism and the ratio thereof in the genes contained in a trace amount in these specimens, which method comprises, in a homogeneous system without a need for any troublesome solid/liquid separation procedures, mixing a first nucleic acid containing the target nucleic acid with a second nucleic acid having a base sequence complementary to a specific domain in the target nucleic acid to perform competitive hybridization, and measuring the extent of the occurrence of the complementary strand substitution between these nucleic acids, thereby distinguishing the identity of the first nucleic acid from the second nucleic acid. In this method, at least two labels capable of mutually transferring energy are introduced into one or both of the first and second nucleic acids and the extent of the energy change caused by the energy transfer between the above labels in association with the complementary strand substitution as described above is measured, thereby evaluating the extent of the complementary strand substitution.

Description

Specification

Nucleic acid identification method and nucleic acid detection kit

The present invention relates to a method for identifying a nucleic acid and a test kit, and more particularly, to a simple method that does not require a solid-liquid separation operation from a sample by determining the presence and proportion of a nucleic acid having a gene mutation or polymorphism from a sample. The present invention relates to a method for identifying a nucleic acid which can be directly detected and quantified in a short time by a simple operation, and a test kit for performing the identification method. Background art

Recent advances in molecular biology have been remarkable, and the decoding of the entire nucleotide sequence of a human gene has become a reality. In addition, data on the relationship between gene polymorphisms and diseases is being accumulated every day, and polymorphism analysis of the entire human gene region is also progressing. Under such circumstances, it will be necessary to accurately detect a large number of gene mutations or polymorphisms in a large number of gene regions (in other words, a single nucleotide mutation) in a short time. This is a very important issue in medical practice.

As a method for detecting such a mutation in a gene, a detection method using an oligonucleotide probe (Proc. Nat 1. Acad. Sci. 80, 278, ( 198 3)), a method using restriction enzyme fragment length polymorphism analysis (RFLP method) (Am. J. Hum. Genet., 69, 210 (1980)), A method for cleaving a single base mismatch in a hybrid of RNA and DNA using ribonuclease (Science 230, 1243 (19895)). Natl. Acad. Sci. 88, 189 (1991), Anal. Bioche m. 1886, 64-4-6 8 (1990)).

However, both methods use specific mutations whose base sequences are known. Since the method is limited to detection and the operation is complicated, it cannot be said that this method is suitable for measuring gene mutations in medical practice.

On the other hand, as a method for detecting unspecified mutations (positions, bases) in a certain region of a target nucleic acid in a specimen, SSCP (single-strand conformation ionpolymorphism) method (Procion conformation polymorphism) is used. Natl. Acad. Sci. 86, 2766 (1989)), DGGE method (Proc. Natl. Acad. Sci. 86, 2332, (19) 8 9)) and the ddF method (Genomics 13, 4 41 (1992)) have been reported.

However, any of the above methods has a problem that the reproducibility is low and the electrophoresis method is used for the detection, so that the method lacks quickness and is not practical. In addition, a method of determining the base sequence of the target nucleic acid by using an automatic sequencer and detecting the mutation can be considered, but it is difficult to detect when the sample is a mixture or the ratio of the mutant nucleic acid is small. It was possible. In recent years, microchip-type measurement technology using a very small amount of pitting technology has been developed, and is expected to be applied to new drug research and gene polymorphism analysis. This method detects mutated nucleic acids by immobilizing tens of thousands of DNA probes on a fine carrier surface and hybridizing with a labeled sample (Nucleic Acids Res. 2Q). 6, 4975-4982 (19998)), which can process a large number of specimens at once, which can save a great deal of labor, but has low reproducibility and special equipment There is a problem when the cost is high because of the use of

Therefore, as a rapid identification method for unspecified mutations in a specific region, the applicant of the present invention has proposed a method of adding an excessive amount of unlabeled standard DNA to a labeled sample DNA and performing cooperative competitive hybridization. In addition, we have developed a method that can detect the presence or absence of a gene mutation or polymorphism regardless of the type of nucleic acid and can easily calculate the abundance ratio, and have already proposed it (hereinafter abbreviated as PCR-PHFA method. International Publication W095 / 0208, Nuc1.Acids.Res.22, 154 (1994)). According to the PCR-PHFA method, it is possible to reliably detect or quantify a mutation or polymorphism gene that is present only in a small amount in a normal gene or even when the type of mutation or polymorphism is unspecified. In fact, it is very effective in detecting nucleic acids having genetic mutations and polymorphisms such as genes related to genetic diseases or cancer-related genes (British J. Hemato 1 ogy 95, 198— 203).

However, the PCR-PHFA method requires complicated solid-liquid separation work after competitive hybridization, and gene mutation or multiplication by simple operations at the forefront medical site. It has not been able to adequately respond to the demand for rapid detection of nucleic acids having a form. In addition, the relationship between genetic mutations and diseases is very complex and diverse, and the rare cases in which a single genetic mutation found at the outset causes disease are relatively rare and unexpectedly many. It is becoming clear that the gene mutations involved in the onset of the disease are evident.To investigate the gene mutations that cause a certain disease, it is necessary to check many mutations for multiple genes without fail. Therefore, development of a nucleic acid detection method that can automatically and easily process a large amount of a sample more easily and quickly is desired.

Recently, Ge1 fand et al., As shown in Fig. 4, labeled the 5'-ends of each of a pair of 13-base pair oligonucleotides with fluorescent substances A and D, and showed that the two strands were double-stranded. In the case where a complex is formed, fluorescence energy transfer occurs.On the other hand, when a competing oligonucleotide is added, the degree of the fluorescence energy transfer changes when recombination with the original duplex occurs. (Proc. Natl. Acad. Sci. USA 96, 61 13-61 18 (1999)). That is, it shows that the difference in stability between the original double-stranded DNA and the double-stranded DNA formed by recombination can be measured by using energy transfer.

However, in this method, there is a label at the 5 'end of each strand of the oligonucleotide that can transfer fluorescent energy, so that When the length of the leotide chain becomes longer, the distance between the two labels becomes longer, and accordingly, the degree of change in energy transfer due to DNA recombination also becomes smaller, which may make detection difficult. Particularly, in order to reliably detect gene mutations and polymorphisms with high sensitivity, it is usually necessary to have a certain chain length, so that sufficient results cannot be obtained. Disclosure of the invention

The present invention has been made in view of the above circumstances, and the presence and the ratio of nucleic acid having a gene mutation or polymorphism can be determined in a short time directly by a simple operation that does not require a solid-liquid separation operation from a sample. An object of the present invention is to provide a nucleic acid identification method that can be accurately detected and quantified and that can be automated, and a test kit for performing the identification method.

According to the present invention, in order to achieve the above object, a first nucleic acid and a second nucleic acid are mixed and subjected to competitive hybridization, and the degree of substitution of complementary strand between the two nucleic acids is measured. Thus, in the method for identifying the identity of the first nucleic acid and the second nucleic acid,

(1) At least one of two types of labels capable of energy transfer to each other is introduced into the 3 ′ end of one strand of the first nucleic acid, which is a double-stranded nucleic acid, and the other strand of the first nucleic acid is The first nucleic acid is labeled by introducing the other label at the 5 'end of the nucleic acid, and the labeled first nucleic acid is mixed with the unlabeled second nucleic acid, which is a single-stranded or double-stranded nucleic acid, and mixed. By performing pettive hybridization and measuring the degree of energy change due to energy transfer between the labels, the degree of displacement of the complementary strand generated between the first nucleic acid and the second nucleic acid can be determined. Measuring, or

(2) Labeled first nucleic acid prepared by introducing one of at least two kinds of labels capable of energy transfer to each other into the first nucleic acid, and label prepared by introducing the other label into the second nucleic acid The second nucleic acid is mixed with the following combinations (a) to (c) to perform competitive hybridization, and the degree of energy change due to the energy transfer between the labels is measured. By doing so, the degree of replacement of the complementary strand generated between the first nucleic acid and the second nucleic acid can be measured.

(1) Both the first nucleic acid and the second nucleic acid are double-stranded nucleic acids, and a labeled first nucleic acid having one label introduced at the 3 ′ end of one strand, and a label introduction of the labeled first nucleic acid A combination with a labeled second nucleic acid in which the other label is introduced at the 5 'end of the strand to be hybridized with the strand,

(Mouth) A labeled first nucleic acid in which one label is introduced into the 3 'end of one strand of the first nucleic acid, which is a double-stranded nucleic acid, and a 5' end of the second nucleic acid, which is a single-stranded nucleic acid A combination with a labeled second nucleic acid in which the other label is introduced,

(C) a labeled first nucleic acid in which one label is introduced into the 5 'end of one strand of the first nucleic acid, which is a double-stranded nucleic acid, and a 3' end of the second nucleic acid, which is a single-stranded nucleic acid And a combination with a labeled second nucleic acid into which the other label has been introduced.

In other words, the PCR-PHFA method proposed by the present applicant can amplify a specific region of a target nucleic acid in a sample to prepare a double-stranded sample nucleic acid, and bind to one of the strands with a solid phase carrier. Competitive hybridization is carried out by adding an excess amount of the sample nucleic acid to a labeled standard nucleic acid comprising a double-stranded nucleic acid having a unique site and a detectable label on the other strand, and as a result, By detecting the reconstituted labeled standard nucleic acid using the detectable label and the label having a site capable of binding to a solid support,

By detecting the degree of substitution of the complementary strand between the sample nucleic acid and the labeled standard nucleic acid, a nucleic acid having a gene mutation or polymorphism is detected. As described above, since this method uses a detectable label and a label having a site capable of binding to a solid phase carrier, a cumbersome solid-liquid separation operation is required, and the operation is simple. It has not been widely adopted in the medical field where quick and accurate detection is required.

In the method of Ge1 fand et al., As shown in Fig. 4, if a pair of oligonucleotides is as short as about 13 base pairs, label D Since energy and label A are relatively close to each other, energy translocation between the labels occurs, but as described above, as the base pair length increases, the distance between label D and label A increases. Since energy transfer between the labels does not occur or is extremely reduced, it is difficult to identify the identity of nucleic acids having gene mutations or polymorphisms using gene fragments of various lengths.

On the other hand, in the above-described identification method of the present invention, at least two kinds of labels capable of energy transfer with each other are used, and the degree of energy change due to the energy transfer between the two labels is measured. Since the degree of displacement of the complementary strand due to the hybridization is measured, the first nucleic acid and the first nucleic acid can be quickly and easily collected without complicated work such as solid-liquid separation. (2) The identity with the nucleic acid can be identified, and both labels are introduced at the 3 'end and the 5' end which are close to each other, so that the degree of displacement of the complementary strand can be accurately and accurately determined. In addition to being able to reliably capture, even if the first nucleic acid or the second nucleic acid is a gene fragment having a long chain, the degree of substitution of the complementary strand can be accurately and accurately measured with good sensitivity at all times, and the identity of the nucleic acids can be determined. Accurate and Ru 3D in what can and this identifies the Joteki.

Therefore, according to the identification method of the present invention, one of the first nucleic acid and the second nucleic acid is used as a sample nucleic acid containing a target nucleic acid, and the other is used as a standard nucleic acid for identifying the identity of the sample nucleic acid. Thus, nucleic acids having mutations or polymorphisms of a large number of genes in a large number of samples can be quickly and accurately detected and quantified, and the conventional complicated solid-liquid separation work is not required. It can be automated with a simple method, and can respond to the demands of the forefront medical field. In this case, even if the amount of nucleic acid having a gene mutation or polymorphism in the sample is extremely small, or if the normal nucleic acid differs from the mutant nucleic acid (wild-type gene and mutant gene) by only one base, it is accurate. It can be detected and quantified by simple operations.

In the present invention, the 5 'end and the 3' end indicate a range within 30 bases from the 5 'end and the 3' end of the nucleic acid chain, respectively. . In this case, energy transfer is more likely to occur closer to the 5 'end and the 3' end, so it is preferably within 10 bases from each end, and most preferably the 5 'end and the 3' end. The end.

Further, the present invention provides a kit for performing the identification method of the present invention,

A sample nucleic acid amplification reagent for amplifying a specific region of a target nucleic acid in a sample to prepare a sample nucleic acid; a standard nucleic acid amplification reagent for preparing a standard nucleic acid for identifying the identity of the target nucleic acid; A reagent for introducing at least two kinds of labels capable of energy transfer in the vicinity of each other to one or both of a sample nucleic acid and a standard nucleic acid; and a nucleic acid inspection kit. To provide

According to this test kit, a nucleic acid having a trace amount of a mutation or a polymorphic gene in a sample can be rapidly and reliably detected by a simple operation according to the identification method of the present invention, and automation can be achieved. , At the forefront of medical care

It is extremely useful in I 5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing a case where the method for identifying a nucleic acid according to the first invention is performed, wherein (A) shows a normal nucleic acid and (B) shows a nucleic acid having a mutation.

2 0

FIG. 2 is a schematic diagram showing a case where the method for identifying a nucleic acid according to the second invention is performed. (A) shows a normal nucleic acid, and (B) shows a nucleic acid having a mutation. 2 is a fluorescence vector showing the state of energy transfer between the two types of labels shown in FIG.

FIG. 4 is an explanatory diagram showing the conventional method of Ge1fand. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail. The method for identifying a nucleic acid according to the present invention comprises the steps of: mixing a first nucleic acid and a second nucleic acid; performing implicit hybridization; and measuring the degree of occurrence of complementary strand displacement. In the method for identifying the identity between the first nucleic acid and the second nucleic acid, at least two labels capable of energy transfer to each other are introduced into one or both of the first nucleic acid and the second nucleic acid. By measuring the degree of energy change due to the energy transfer between the labels accompanying the displacement of the complementary strand, the degree of the displacement of the complementary strand is measured. — At least energy transfer in close proximity to each other instead of the two labels in the PHFA method

10 Two types of labels (for example, a donor label that generates fluorescence upon excitation and an receptor label that absorbs the fluorescence) are introduced into the first nucleic acid and the Z or the second nucleic acid, and a competitive hybridization is performed. (Complementary strand displacement reaction).

In the first method of the identification method of the present invention, one of the two types of labels is introduced as a double-stranded nucleic acid at the 3 ′ end of one strand of the first nucleic acid, and the other strand of the first nucleic acid is The other label is introduced into the 5 ′ end of the nucleic acid to prepare a labeled first nucleic acid, and the labeled first nucleic acid is mixed with a double-stranded or single-stranded unlabeled second nucleic acid to form a compound. The first nucleic acid can be obtained by performing pettive hybridization and measuring the degree of change in energy transfer between the labels.

The degree of substitution of the complementary strand generated between 20 and the second nucleic acid is measured to discriminate the identity between the first nucleic acid and the second nucleic acid.

To explain the principle, as shown in Fig. 1 (A), two types of labels D and A capable of binary energy rearrangement (donor label D that generates energy and energy from the donor label are absorbed) Axceptor label A)

2 5 is introduced into the 3 ′ end of one strand and the 5 ′ end of the other strand of the first nucleic acid, which is a double-stranded nucleic acid, to prepare a double-stranded labeled first nucleic acid 1. that _c the labeled first nucleic acid 1 2 kind of labeled D, a energy transfer occurs in some of the state is close, Enerugi one generated from the donor label D is absorbed by the § acceptor labeled a in donor label D Energy drops However, label A, which has absorbed energy, generates energy and energy translocation occurs.

Next, the unlabeled second nucleic acid 2 (which is a double-stranded nucleic acid in FIG. 1 but may be a single-stranded nucleic acid) which is homologous to the labeled first nucleic acid 1 is mixed and denatured, and then annealed.

Competitive hybridization is performed by 5 ringing: a complementary strand displacement reaction occurs, and as shown in the figure, a nucleic acid in which two types of labels D and A are in close proximity is obtained. Upon dilution, a synthetic nucleic acid having only the donor label D or only the acceptor label A results, resulting in a reduced proportion of nucleic acids that undergo energy translocation.

10 On the other hand, as shown in FIG. 1 (B), a similar hybrid-stranded first nucleic acid 1 is mixed with an unlabeled second nucleic acid 2 ′ having a mutation X to perform a competitive hybridization. Since the complementary strand displacement reaction does not occur, the proportion of nucleic acids that cause energy translocation does not change (as is).

Therefore, by measuring the degree of energy transfer shown in FIGS. 1 (A) and 1 (B), the degree of displacement of the complementary strand generated between the first nucleic acid 1 and the second nucleic acid 2 or 2 ′ is determined. Thus, the identity between the first nucleic acid 1 and the second nucleic acid 2 or 2 ′ can be identified. One of the first nucleic acid 1 and the second nucleic acid 2 or 2 ′ is used as a sample nucleic acid containing the target nucleic acid, and the other is used as a standard nucleic acid for identifying the identity of the sample nucleic acid.

According to the formula, the presence / absence of the gene mutation in the sample nucleic acid and the ratio thereof can be measured.

In the example shown in FIG. 1, a nucleic acid having a known mutation as the first nucleic acid 1 was prepared, and the first nucleic acid 1 having this mutation and the second nucleic acid 2 or 2 ′ were mixed to form a competitive nucleic acid. Performing a hybrid session

According to 25, the presence or absence of the mutation in the gene in the second nucleic acid 2 or 2 ′ and the ratio thereof can be similarly detected.

Further, the second method of the identification method of the present invention comprises the steps of: combining a label first nucleic acid prepared by introducing at least one of two kinds of labels capable of energy transfer with each other into the first nucleic acid; The labeled nucleic acid prepared by introduction into the second nucleic acid The two nucleic acids are mixed with the following combinations (a) to (c) to perform a competitive hybridization, and the degree of energy change due to energy-translocation between the labels is measured. The degree of substitution of the complementary strand generated between the first nucleic acid and the second nucleic acid is measured to discriminate the identity between the first nucleic acid and the second nucleic acid.

(A) Both the first nucleic acid and the second nucleic acid are double-stranded nucleic acids, and a labeled first nucleic acid in which one label is introduced into the 3 ′ end of one strand, and a label of the labeled first nucleic acid A combination of the introduced strand and a labeled second nucleic acid in which the other label is introduced at the 5 'end of the strand to be hybridized.

(Mouth) A labeled first nucleic acid in which one label is introduced into the 3 'end of one strand of the first nucleic acid which is a double-stranded nucleic acid, and a 5' end of the second nucleic acid which is a single-stranded nucleic acid In combination with a labeled second nucleic acid in which the other label is introduced.

(C) a labeled first nucleic acid in which one label is introduced into the 5 'end of one strand of the first nucleic acid, which is a double-stranded nucleic acid, and a 3' end of the second nucleic acid, which is a single-stranded nucleic acid In combination with a labeled second nucleic acid in which the other label is introduced.

The principle is explained using the case (a) above as a model. As shown in Fig. 2 (A), two energy-transferable labels D and A are attached to one strand of the first nucleic acid 3 One type is introduced into each of the 3 ′ end and the 5 ′ end of the other strand of the second nucleic acid 4 to prepare a labeled first nucleic acid 3 and a labeled second nucleic acid 4. These labeled nucleic acids 3 and 4 have a label on only one of the two strands. That is, since both have only one type of label, energy transfer does not occur. 2 Competitive hybridization is performed by mixing and denaturing nucleic acid 4 and annealing. This causes a complementary strand displacement reaction to generate a double-stranded nucleic acid in a state where the two types of labels D and A are in close proximity, and energy transfer occurs.

On the other hand, as shown in FIG. 2 (B), the two types of energy transferable labels D and A are replaced with the 3 ′ end of one strand of the first nucleic acid 3 and the second nucleic acid 4 ′ having a mutation X. One by one at the 5 'end of the other strand of Prepare labeled second nucleic acids 4 ′ and 4 ′. Competitive hybridization is performed by mixing and denaturing the labeled first nucleic acid 3 and the labeled second nucleic acid 4 ', followed by annealing. In this case, since no complementary strand displacement reaction occurs, no nucleic acid in which the two types of labels D and A are close to each other is generated, and no energy transfer occurs.

Therefore, by measuring the degree of energy transfer shown in FIGS. 2 (A) and (B), the degree of substitution of the complementary strand generated between the first nucleic acid 3 and the second nucleic acid 4 or 4 ′ can be determined. As a result, the identity between the first nucleic acid 3 and the second nucleic acid 4 or 4 ′ can be identified. Then, one of the first nucleic acid 3 and the second nucleic acid 4 or 4 ′ is used as a sample nucleic acid containing the target nucleic acid, and the other is used as a standard nucleic acid for identifying identity with the sample nucleic acid. It can measure the presence or absence of the gene mutation in the sample nucleic acid and its ratio.

In the example shown in FIG. 2, a nucleic acid having a known mutation was prepared as the first nucleic acid 3, and the first nucleic acid 3 having the mutation was mixed with the second nucleic acid 4 or 4 ′, and The presence or absence of the mutation in the gene in the second nucleic acid 4 or 4 ′ and the ratio thereof can be similarly detected by performing the competitive hybridization.

In the case of the above (Mouth) and (C) in the second method, one of the nucleic acids of the first nucleic acid 3 and the second nucleic acids 4 and 4 ′ in FIG. 2 is a single-stranded nucleic acid. The principle is the same as that of the double-stranded nucleic acid shown in FIG.

As described above, the nucleic acid identification method of the present invention directly measures the degree of change in energy transfer between at least two kinds of labels capable of performing energy transfer. It is possible to detect the presence or absence of the nucleic acid having the gene mutation or polymorphism in the sample and the ratio thereof quickly and reliably by a simple operation without the necessity. In addition, since both labels are introduced at the 3 'end and the 5' end which are close to each other, even when the degree of substitution of the complementary strand is small, a sufficient energy change that can be easily detected is generated, and correct In addition, the degree of substitution can be accurately and reliably measured with good sensitivity even if the first or second nucleic acid is a gene fragment having a long chain.

In the method for identifying a nucleic acid according to the present invention, one of the first nucleic acid and the second nucleic acid is used as a sample nucleic acid containing a target nucleic acid, and the other is used as a standard nucleic acid for identifying identity with the sample nucleic acid. It is preferably used for detecting the presence or absence of a nucleic acid having a gene mutation or polymorphism and its ratio. In this case, first, a specific region of the target nucleic acid in the sample is amplified to prepare a sample nucleic acid, and a standard nucleic acid having the same region as the specific region of the target nucleic acid is prepared. At this time, the target nucleic acid to be detected includes cancer-related genes, genes related to genetic diseases, virus genes, bacterial genes, and polymorphisms called disease risk factors. And the like.

Here, among the target nucleic acids, examples of the cancer-related gene include a k-ras gene, an N-ras gene, a p53 gene, a BRCA1 gene, a BRCA2 gene, and an APC gene. Genes related to genetic diseases include various inborn errors of metabolism. Examples of viruses and bacterial genes include hepatitis C virus and hepatitis B virus. Genes that show polymorphism are genes that have different nucleotide sequences depending on individuals that are not directly related to the cause of the disease, such as HLA (Human Leukocyte Antigen) and blood type genes. Alternatively, there are genes that are considered to be involved in the onset of hypertension, diabetes and the like. These genes are usually present on the chromosome of the host, but may be encoded by the mitochondrial gene.

Examples of such specimens containing the target nucleic acid include pathogens such as bacteria and viruses, blood, saliva, tissue debris isolated from living bodies, and excrement such as manure. In addition, when performing prenatal diagnosis, fetal cells present in amniotic fluid or a part of dividing egg cells in a test tube can be used as a sample. In addition, these samples may be centrifuged directly or as necessary. After being concentrated as a sediment by a separation operation or the like, it is possible to use, for example, an enzyme treatment, a heat treatment, a surfactant treatment, an ultrasonic treatment, or a cell treatment that has been subjected to cell destruction treatment by a combination thereof in advance. it can. In this case, the cell destruction treatment is performed for the purpose of revealing the DNA derived from the target tissue. The specific method of the cell disruption treatment is as follows: PCR protocol / less Academic Press Ink pl 4, p 352 (1990) (PCRPROTOCOLSAcademic Press Inc., pl 4 , P 352 (1990)), etc., according to known methods described in literatures. It is preferable that the total amount of DNA in the sample is about 1 to 10 g, but amplification can be sufficiently performed with 1 μg or less. ·

In this case, the sample nucleic acid and the standard nucleic acid, that is, the first nucleic acid and the second nucleic acid are obtained by a known PCR (Polymerase Chain Reaction) method, L-R (lgasechain Reaction) ¾ 3 SR (Self-sustained S) prepared by the equence Rep 1 ication) method, SDA (Strand D isp 1 acement Amp 1) method, etc. (Manak, DNA Probes 2nd Editionp 255 to 291, Stokton Press) (1993)), PCR is particularly preferred.

Here, the PCR method will be described in more detail. The primer for amplifying the sample nucleic acid and the standard nucleic acid (the first nucleic acid and the second nucleic acid) is, as long as the sample nucleic acid and the standard nucleic acid are present, a primer extension. A gene amplification reaction based on the reaction occurs. In this case, the extension reaction of the primers was carried out with four or five nucleotide triphosphates (deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and thymidine triphosphate). The reaction is carried out by incorporating an acid or dextrinidine triphosphate (a mixture of these as dNTP, or a mixture thereof)) into the primer as a substrate. When performing this extension reaction, an amplification reaction reagent containing the above-mentioned unit nucleic acid and nucleic acid elongation enzyme is usually used to amplify the nucleic acid chain. In this case, the nucleic acid elongation enzyme is E. coli DNA polymerase. Any DNA polymerase, such as Klenow fragment of E. coli DNA polymerase I, or DNA polymerase 4 can be used. Particularly, Taq DNA polymerase, T It is preferable to use a thermostable DNA polymerase such as th DNA polymerase and Vent DNA polymerase, which eliminates the need to add a new enzyme for each cycle. This makes it possible to automatically repeat the cycle, and furthermore, it is possible to set the annealing temperature to 50 to 60 ° C, thereby improving the specificity of the target base sequence recognition by the primer. Rapid and specific gene amplification (For details, refer to JP-A-11-314965 and JP-A-252230).

Further, when performing this reaction, oil can be added to prevent evaporation of water in the reaction solution. In this case, the oil may be any oil that can be distributed with water and has a lower specific gravity than water, and specific examples include silicone oil and mineral oil. In addition, some gene amplification devices do not require such a medium, and a primer extension reaction can be performed using such a gene amplification device.

In this way, by repeating the extension reaction using the above-described nucleic acid amplification primer, the target nucleic acid in the sample can be efficiently amplified and the sample nucleic acid can be prepared in a large amount. In addition, a large amount of a standard nucleic acid whose identity with the target nucleic acid is to be identified can be similarly prepared. Specific methods such as conditions for carrying out the gene amplification reaction are described in Experimental Medicine, Yodosha, 8, No. 9 (1990), and PCR Technology Stockton. Pressing can be performed according to a known method described in a literature such as a press (PCR Technology Locktonpress (1989)).

In addition, without using gene amplification, natural enzymes can be The nucleic acid may be directly cut out enzymatically, or the amplified normal nucleic acid may be added to a vector selected from a plasmid vector, a phage vector, or a plasmid and phage chimera vector. It can also be prepared in large quantities by integration into a host such as Escherichia coli, Bacillus subtilis, or any other proliferable host such as yeast (gene Cloung). Further, in some cases, it can be prepared by chemical synthesis. Examples of chemical synthesis include the ester method and the phosphite method. These methods are based on a liquid phase method or a solid phase synthesis method using an insoluble carrier. 392)), a large amount of single-stranded DNA can be prepared, and then annealing can be performed to prepare double-stranded DNA.

One of the sample nucleic acid and the standard nucleic acid thus prepared in a large amount as described above is used as the first nucleic acid, and the other is used as the second nucleic acid, and one or both of the first and second nucleic acids are used as energy. Introduce at least two transposable labels.

Here, the energy transfer between the labels in the present invention means that at least two types of labels, a donor label for generating energy and an axebuta label for absorbing energy generated from the donor label, are mutually separated. The transfer of energy from a donor label to an acceptor label when in close proximity. For example, when the two types of labels are fluorescent labels, the fluorescent label generated by exciting the donor label is absorbed by the receptor label, and the force for measuring the fluorescence emitted by the receptor label, or the fluorescence generated by exciting the donor label is generated. The quenching of the donor label caused by the absorption of the fluorescence by the receptor label can be measured (PCRM ethodsandapp 1 ications 4, 35 7 — 3 62 (1995), Nature). Biotechnology 16, 49-53 (1998). Energy transfer may occur even if the fluorescence wavelength of the donor label and the absorption wavelength of the acceptor label do not overlap, and such energy transfer is also included in the present invention. The at least two kinds of labels are not particularly limited as long as they are capable of energy transfer in a state close to each other. Among them, a fluorescent substance and a delayed fluorescent substance are preferable, and in some cases, a chemiluminescent substance, Bioluminescent materials can also be used. Such label combinations include fluororesin and its derivatives (for example, fluorescein isothiosinate) and rhodamine and its derivatives (for example, tetramethyl rhodamin isothiosinate, tetramethyl rhodamine). 5 — (and — 6-) hexanoic acid acid, etc.), fluorescein and dabsir, and any combination can be selected from these (Nonisotopic DNAP robe Ίechniques. Academic Press 1992) No.

As a method for introducing the two kinds of labels into one or both of the sample nucleic acid and the standard nucleic acid (the first nucleic acid and the second nucleic acid), a general method of introducing a label into a nucleic acid may be employed. it can. For example, a method for directly introducing a labeled substance into a nucleic acid (Biotechniques 24, 484-489 (1998)), a DNA polymerase reaction or an RNA polymerase reaction. Method of introducing labeled mononucleotides (Science 238, 336-33341 (19897)), and performing a PCR reaction using a primer into which the labeled substance has been introduced. (PCRM ethodsand Applications 2, 34-40 (1992)).

In this case, the position where the label is introduced into the sample nucleic acid and the standard nucleic acid (the first nucleic acid and the second nucleic acid) is the position where energy transfer occurs or disappears due to the complementary strand displacement reaction, that is, the 3 ′ of the nucleic acid strand. Must be end and / or 5 'end. Specifically, it shows a range within 30 bases from the 5 'end and the 3' end of the nucleic acid strand, respectively.The closer the labels are, the more likely it is for the energy to transpose. It is within 1 ° from each terminal, most preferably 5 ′ terminal and 3 ′ terminal. Here, when a large number of labels are introduced into the base portion that hybridizes with the complementary strand, Since the substitution of about a group may become undetectable, it is preferable to introduce only into the end of each nucleic acid strand. For example, one of the two types of labels is introduced at the 5 'end (3' end) of one nucleic acid strand, and the 3 'end (5' end) of the other nucleic acid strand complementary thereto is introduced. If the other label is introduced into the second strand, both nucleic acid strands undergo energy transfer or disappear by the complementary strand displacement reaction without affecting the hybridization reaction.

Specifically, in order to prepare a nucleic acid strand having a label at the 5 'end, a method in which a PCR reaction is performed using a primer having a label at the 5' end (PCRM ethodsand Applications 2, 34) -40 (1992)) or a method in which a linker having a label introduced at the 5 'end and an arbitrary nucleic acid strand are bound by ligase (Nuc 1 eic Acids Res. , 9 2 2 — 9 2 3 (1 9 9 7)).

On the other hand, in order to prepare a nucleic acid strand having a label at the 3 ′ end, a linker having a label introduced at the 3 ′ end and an arbitrary nucleic acid strand are prepared in the same manner as when the label is introduced at the 5 ′ end. There is a method of binding ligase. When the nucleic acid chain is RNA instead of DNA, or when the 3 ′ end of DNA is RNA, it is generated by selectively opening the sugar (ribose) portion of the RNA at that end. Labeling can also be carried out using an aldehyde group: In addition, the labeled mononucleotide triphosphate can be used to terminate the nucleic acid strand by the action of terminal hydroxy nucleotidyl transferase. It is also possible to introduce a marker at the 3 'end (Biotechniques 15, 486-496 (1993)).

When the sample nucleic acid and the standard nucleic acid (the first nucleic acid and the second nucleic acid) are relatively short nucleic acid chains of 100 bases or less, the labeled nucleic acid can be prepared by direct chemical synthesis. (Nucleic A cids Res. 16, 2659-2669 (1988), Bioconjug. Chem. 3, 85-87 (1992)) _c Next, competitive hybridization is performed using the sample nucleic acid and the standard nucleic acid (the first nucleic acid and the second nucleic acid) into which the label has been introduced as described above.

In this case, the competitive hybridization in the present invention refers to between a double-stranded nucleic acid having a homologous base sequence and a single-stranded nucleic acid, or between a double-stranded nucleic acid having a homologous base sequence and a double-stranded nucleic acid. Is a competitive nucleic acid strand displacement reaction that takes place between denaturing and annealing single- and double-stranded nucleic acids, or multiple double-stranded nucleic acids. First, if the sample nucleic acid and Z or the standard nucleic acid (first nucleic acid and / or second nucleic acid) are double-stranded, they need to be denatured. A force-based method is preferred. The time when the sample nucleic acid and the standard nucleic acid are mixed may be either immediately before the denaturation or after the denaturation.

Furthermore, it is necessary to adjust the salt concentration in the reaction solution to be optimum, which largely depends on the chain length. In general, in hybridization, SSC (20 XSSC: 3 M sodium chloride, 0.3 M sodium citrate) and SSPE (20 XSSPE: 3.6 M sodium chloride) are used. , 0.2 M sodium phosphate, 2 mM EDTA), and these solutions can be used after being diluted to a suitable concentration in the identification method of the present invention. If necessary, an organic solvent such as dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) can be added.

The temperature conditions of the competitive hybridization are appropriately set according to the length and base sequence of the nucleic acid to be hybridized, but are usually in the range of 98 to 50 ° C for 3 to 10 minutes. The temperature can be reduced at a rate of 1 ° C, more preferably in the range of 98-70 ° C, at a rate of 1 ° C in 10 minutes.

The method for identifying a nucleic acid according to the present invention comprises at least two kinds of targets as described above. By subjecting the sample nucleic acid and the standard nucleic acid (the first nucleic acid and the second nucleic acid) into which the knowledge has been introduced to thermal denaturation or alkaline denaturation, a competitive hybridization is performed to gradually lower the temperature from a high temperature. A nucleic acid having a gene mutation or a polymorphism is detected by performing annealing and measuring the degree of change in energy transfer between the labels.

In this case, a nucleic acid having a completely complementary base sequence forms a double strand more preferentially than a nucleic acid having a gene mutation or a polymorphism, and accordingly, the energy change between the labels. The degree of change in energy due to the displacement, that is, the degree of change in energy transposition that occurs or disappears due to the complementary strand displacement reaction, is measured using an arbitrary detector, so that gene mutation can be detected. Alternatively, the presence or absence of the nucleic acid having the polymorphism and the ratio thereof can be detected. For example, when fluorescence energy translocation is used for detection, gene mutation or multiple mutations can be measured by measuring the fluorescence spectrum at a specific wavelength with a spectrofluorometer, fluorescence blade reader, or the like. The presence or absence of a nucleic acid having a type can be easily detected.

Specifically, at least two types of labels that can be energy-transferred in close proximity to each other (for example, a donor label that emits fluorescence by excitation and an acceptor label that absorbs the fluorescence) are used. One of the two labels is introduced into the 3 ′ end of one strand of the first nucleic acid, which is a double-stranded nucleic acid, and the other label is attached to the 5 ′ end of the other strand of the first nucleic acid. In addition to preparing the labeled first nucleic acid, a double-stranded or single-stranded unlabeled second nucleic acid is mixed with the labeled first nucleic acid to perform competitive hybridization, and the above-described labeling is performed. By measuring the degree of change in the energy transfer, the degree of substitution of the complementary strand generated between the first nucleic acid and the second nucleic acid is measured to determine the identity between the first nucleic acid and the second nucleic acid. The identity of the standard nucleic acid and the sample nucleic acid, Presence and proportion thereof of nucleic acids having a mutation or polymorphism of the child can be a detection child a.

Also, the labeled first nucleic acid prepared by introducing the one label into the first nucleic acid and the labeled second nucleic acid prepared by introducing the other label into the second nucleic acid are as follows. The above-mentioned first nucleic acid is obtained by mixing in a combination of (a) to (c), performing a competitive hybridization, and measuring the degree of energy change due to energy-transposition between the labels. The degree of displacement of the complementary strand generated between the first nucleic acid and the second nucleic acid is measured to identify the identity between the first nucleic acid and the second nucleic acid, that is, the identity between the standard nucleic acid and the sample nucleic acid, and It is possible to detect the presence or absence of a nucleic acid having a gene mutation or polymorphism and its ratio.

(A) the first nucleic acid and the second nucleic acid are both double-stranded nucleic acids, and a labeled first nucleic acid having one label introduced at the 3 ′ end of one strand; and a label introduction of the labeled first nucleic acid. Combination with a labeled second nucleic acid having the other label introduced at the 5 'end of the strand to be hybridized with the strand.

(Mouth) a labeled first nucleic acid in which one label is introduced into the 3 'end of one strand of the first nucleic acid, which is a double-stranded nucleic acid, and a 5' end of the second nucleic acid, which is a single-stranded nucleic acid In combination with a labeled second nucleic acid in which the other label is introduced.

By using a combination of two or more kinds of energy transferable labels, it is possible to simultaneously detect nucleic acids having mutations or polymorphisms of a plurality of genes in one reaction vessel. It is.

Furthermore, in the method for identifying a nucleic acid of the present invention, the degree of energy transfer is significantly changed according to the ratio of the wild-type gene and the mutant gene in the sample. If a standard curve is prepared in advance and determined, the ratio of the wild-type gene to the mutant gene can be easily known.

Next, the test kit for nucleic acid identification of the present invention is a test kit for detecting the presence and proportion of nucleic acid having a gene mutation or polymorphism according to the above-described identification method of the present invention. As a result, a sample nucleic acid is prepared from a specific region of the target nucleic acid of the sample that has been subjected to pretreatment such as cell destruction treatment. A standard nucleic acid having a base sequence complementary to the sample nucleic acid is prepared, and the sample nucleic acid and the standard nucleic acid are used as the first nucleic acid and the second nucleic acid, respectively, and at least one of which can be energy-transferred to one or both of them. By introducing two types of labels, mixing the sample nucleic acid and the standard nucleic acid, performing a competitive hybridization, and measuring the degree of change in energy transfer between the labels, the complementation is achieved. It measures the extent to which strand displacement has occurred.

In this case, a sample nucleic acid amplification reagent for amplifying a specific region of the target nucleic acid in the sample to prepare a sample nucleic acid, and a standard for preparing a standard nucleic acid for identifying the identity of the sample nucleic acid and the target nucleic acid Identification of the nucleic acid of the present invention by combining a nucleic acid amplification reagent and a reagent for introducing at least two kinds of labels capable of energy transfer into one or both of a sample nucleic acid and a standard nucleic acid, And a quantitative inspection kit.

Further, the cell disrupting reagent for sample pretreatment, the washing solution for washing the amplification reaction product, the oil for preventing the evaporation of the water in the reaction solution, and two kinds of labels described in the above-described nucleic acid identification method of the present invention. A reagent or the like for measuring the degree of change in energy transfer between the two can be used, and in combination therewith, the inspection kit of the present invention can be used.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples.

CF i 'R (cysticfibrosis transmembrane conductance regulator), the causative gene of cystic fibrosis (cysticfibrosis). In order to detect the Δ508 and the Δ508 mutants, labeled and unlabeled oligonucleotides were synthesized by the following method.

First, the wild-type and Δ508 mutant oligonucleotides were used as templates, and the following pair of primers (CF10 ETP, CF10 ETM; Amersham Pharmacia Biotech) Polymerase using A chain reaction (PCR) was performed.

Brima I

C F 10 E T P:

SEQ ID NO: 1 in Sequence Listing

0 — ccattaaaga aaatatcatc—

C F 1 0 Ε Τ Μ ：

SEQ ID NO: 2 in Sequence Listing

5 — atattcatca taggaaacac— ό

The PCR was performed according to a conventional method [denaturation: 30 sec at 94 ° C., annealing: 54. The cycle of 30 sec at C and extension: 6 sec at 72 ° C] was repeated 35 times. After the reaction, the PCR reaction solution was measured by 3% agarose gel electrophoresis. In the wild type PCR reaction solution, a band was detected at a length of 44 bases. In the PCR reaction solution of the Δ508 mutant, a band was detected at a length of 41 bases.

Preparation of labeled and unlabeled oligonucleotides

At the 5 'end of the labeled oligonucleotide, an amino group was introduced using TFA-amino link CE phosphoramidite (manufactured by PerkinElmer Japan), and TAMRA was added. — Using a NHS (Perkin-Elmer Japan), a TAMRA group (Tetramethyllodin-1-5— (and—6—) hexanoyctic acid) was fluorescently labeled at the 5 ′ end.

On the other hand, an amino group was added to the 3 ′ end of the wild-type and Δ508 mutant oligonucleotide using a 3′-amino-modified C7CPG500 (Glen Research). Then, fluorescein isothiocyanate (FITC; Dojindo Laboratories) was fluorescently labeled at the 3 'end.

Oligonucleotides into which these fluorescent labels (FITC, TAMRA) were introduced were purified using high performance liquid chromatography (HPLC) and used as labeled oligonucleotides. On the other hand, oligonucleotides with amino groups introduced were purified using polyacrylamide gel electrophoresis. And used as unlabeled oligonucleotide. Labeled and unlabeled oligonucleotides are named as follows and are shown together with their nucleotide sequences.

C F 1 O W S N 1 -N H, (wild-type unlabeled coding strand): SEQ ID NO: 3 in Sequence Listing

5 o ― H ,, N ― ccattaaaga aaatatcatc tttggtgttt cctatgatga a t at— ό

C F 10 WSN 1 — TAMRA (wild-type labeled coding strand): SEQ ID NO: 3

o ― Γ A ivl R A ― ccattaaaga aaatatcatc tttggtgttt it) cctatgatga a t at— 3

CF 1 OWSN ₂ - NH 2 (complementary strand of unlabeled Cody ring chain wild-type):

SEQ ID NO: 5 in Sequence Listing

ΰ ichi atattcatca taggaaacac caaagatgat attttcttta l 5 atgg— NH ₂ ― 3 '

CF10WSN2-FITC (complement of wild-type labeled coding strand):

SEQ ID NO: 5 in Sequence Listing

5-atattcatca taggaaacac caaagatgat attttcttta

20 atgg-F I T C-3 '

CF 1 OMSN 1 — NH ₂ (unlabeled coding strand of Δ508 mutant):

SEQ ID No. 4

ΰ — H. — ccattaaaga aaatatcatc ggtgtttcct

25 atgatgaata t— 3 '

C F 1 O M S N 1 -T AM RA (labeled coding strand of Δ508 mutant):

SEQ ID NO: 4 in Sequence Listing

b — 1 AMRA — ccattaaaga aaatatcatc ggtgtttcct at ga t gaata t— 3 '

CF10MSN2-NH, (complement of unlabeled coding strand of Δ508 mutant):

SEQ ID NO: 6 in Sequence Listing

o ― atattcat ca t aggaaacac c ga t gatat t tt c t ttaat g g ― N H 3,

CF10MSN2—FITC (complement of labeled coding strand of Δ508 mutant):

SEQ ID NO: 6 in Sequence Listing

o ― atat tcat ca t aggaaacac c ga t gatat t t a a t g g F I T C-3 '

(Example 1)

The state of the energy translocation between the two labels was examined by measuring the fluorescence spectrum. First, a double-stranded DNA was prepared using the above-mentioned labeled and unlabeled oligonucleotide by the following method.

Preparation of double-stranded DNA

Wild-type labeled oligonucleotide CF 10 WSN 1 — Dissolves TAMRA (30 nmol) and CF 10 WSN 2-FITC (30 nmo 1) (10 mM MTris-HC 1 p . H 8 0 5 0 m MN a C 1 1 m MEDTA l O ng / μ Ι herring DNA) was dissolved in 1 0 0 mu 1, di - N'anpu PCR system 9 6 0 0 (Pas one rk (p _e rkin The temperature was lowered continuously to 98 ° C and 60 ° C over 4 hours using a commercial product (Elmer), and the resulting mixture was subjected to arranging. The wild-type labeled double-stranded DNA (A) described below was used. Was prepared.

Wild-type labeled double-stranded DNA

(A) C F I O W S N 1-T A M R A = C F 1 0 W S N 2-F I T C

5 '— TAMRA-c cattaaaga aaatatcatc tt t ggtgt ttcct atgat ga atat— 3 3 — FITC one ggtaat ttct tttatagtag aaac cacaaa gga t ac tact tata— 5

Similarly, the above-mentioned labeled oligonucleotides and unlabeled oligonucleotides were combined to prepare the following double-stranded DNAs (B) to (D).

5 The base sequences of these double-stranded DNAs (B) to (D) are shown below.

A508 Mutant label Double-stranded DNA

(B): C F 10 M S N 1-T A M R A = C F 10 M S N 2-F I T C

o — T A M R A — c cat taaaga aaatat cat c ggt gtt t cc t i o a t gat gaata t— ύ

3 '— FITC-ggtaat t t ct tttatagtag ccacaaagga tac tact tat a— 5'

Wild-type unlabeled double-stranded DNA

(C): CF 10 0 WSN 1-NH ₂ = CF 10 0 WSN 2-FITC l 5 5 — NH 2 ― c cattaaaga aaatat cat c tt tggt gt ttcct at gat ga atat— 3

3 — F I T C i ggtaattt c t t t t atagtag aaaccacaaa gga t actac t t a t a— 5

Δ508 Mutation-labeled unlabeled double-stranded DNA

20 (D): CF 10 MSN 1-NH ₂ = CF 10 MSN 2-FITC

5 — NH ₂ ― ccat taaaga aaatatcatc ggt gt ttccta tga t gaata t— 3 '

3 one F I T shi one ggtaattt c t t t tatagtag ccacaaagga t ac t ac t tat a— 5 '

25 Fluorescence spectrum measurement

Dissolve the solution (10 m) in the double-stranded DNA solution 71 of each of (A) to (D) above.

M Tris — H C 1 H 8.0, 50 mM M Na Cl, ImM

Add EDTA, l Onng (I herring DNA) 4 13 / il and excite using a spectrofluorometer (Shimadzu RF-500) The fluorescence spectrum at a wavelength of 494 nm was measured. The results are shown in Figure 3. From the results in Fig. 3, (A) and (B) show that energy-translocation occurs because the two types of energy transferable fluorescent labels FITC and TAMRA are in close proximity, and the fluorescent label is FITC-type ( Compared to (C) and (D), it was recognized that the fluorescence intensity by FITC was clearly reduced. In this case, only slight fluorescence of TAMRA due to the energy transfer of (A) and (B) was observed.

(Example 2)

Next, the Δ508 mutant was detected using an unlabeled oligonucleotide. First, as No. 1, (A) wild-type labeled double-stranded DNA (CF 10 WSN 1-T AMR A = CF 10 WSN 2-FITC) prepared in Example 1 Type-unlabeled DNA [CF 10 WSN 1-NH, (30 nmo 1) and CF 10 WSN 2 — NH ₂ (30 nmo 1)] and 2 XPHFA solution (6 XSSC, 20 mM Rris — HC 1 pH 8.0, 2 mM EDTA, 20 ng / μ1 herring DNA) 501 was added, and finally, sterile water was added to make the whole 1001. The solution using a GeneAmp PCR system 9 6 0 0 (Parkinson emissions Elmer one (p _e rkin _E lmer) Co.), 9 8. C for 10 minutes, heat denature the DNA, and then reduce the temperature from 98 ° C to 68 ° C at a rate of 1 ° C to reduce the complementary strand displacement reaction (competitive hybridization). ).

Solution obtained in 70/1 solution (10 mM MTris-HC 1 pH 8.0, 50 mM NaCl, 1 mM EDTA, 10 ng / μ1 herring DNA) 3 5 0 xl was prepared and the fluorescence spectrum at an excitation wavelength of 494 nm was measured. Table 1 shows the fluorescence intensity at 517 nm.

In addition, (A) 3 nmol of wild-type labeled double-stranded DNA (CF 10 WSN 1 — T AMR A = CF 10 WSN 2 — FITC) prepared in Example 1 as No. 2 0 8 Mutant unlabeled DNA [CF 10 0 MSN 1 — NH- (30 nmo 1) and CF 10 MSN 2 —NH ₂ (30 nmo 1)] were used in the same manner as in No. 1 except that I did a fib.

A solution (10 mM MTris—HC 1 pH 8.0, 50 mM NaCl, 1 mM EDTA, lOng 1 herring DNA) 3.501 was added to the obtained reaction solution (701). The fluorescence spectrum at an excitation wavelength of 494 nm was measured. Table 1 shows the fluorescence intensity at 517 nm.

3 (B) Δ508 mutant-type labeled double-stranded DNA prepared in Example 1 as No. 3 (CF10MSN1-TAMRA = CF10MSN2-F

1 TC) 3 nmol plus wild type unlabeled DNA [CF 10 WSN 1-NH, (30 nmo 1) and CF 10 WSN 2-NH ₂ (30 nmo 1)]. In the same manner as in 1, a complementary strand displacement reaction (competition) was performed.

Solution obtained in 70 μl of the obtained reaction solution (1 ° m MT ris — HC 1 pH 8.0, 50 mM NaCl, 1 mMEDTA, 10 ng / ^ 1 herring DNA) 3 5 0 xl was measured and the fluorescence spectrum at an excitation wavelength of 494 nm was measured. Table 1 also shows the fluorescence intensity at 517 nm.

In addition, as the No. 4, (B) Δ508 mutant-labeled double-stranded DNA prepared in Example 1 was used as No. 4 (CF10MSN1—TAMRA = CF10MSN

2 - FITC) to 3 nmol Δ 5 0 8 mutant unlabelled DNA [CF 1 0 MSN 1 - NH, (3 0 nmo 1) and _{CF 1 0 MSN 2 - NH 2} (3 0 nmo 1)] was added A complementary strand displacement reaction (competitive hybridization) was carried out in the same manner as in No. 1 except for the above.

Solution obtained in 70 μl of the obtained reaction solution (10 mM MTris — HC 1 PH 8.0, 50 mM NaCl, 1 mM EDTA, lOng / μ1 herring DNA) 350 1 and the fluorescence spectrum at an excitation wavelength of 494 nm was measured. Table 1 shows the fluorescence intensity at 517 nm. I will write it together.

table 1

From the results in Table 1, when No. 1 and No. 2 are compared, No. 1 has 10 equivalents of 10 (A) complementary to each strand of the wild-type labeled double-stranded DNA. In the case of performing a complementary strand displacement reaction with the addition of wild-type unlabeled DNA, No. 2 is 10 equivalents of Δ508 non-labeled DNA to the wild-type labeled double-stranded DNA of (A). Is added to perform a complementary strand displacement reaction. In No.1, the nucleotide sequences of labeled and unlabeled DNA are exactly the same.

10. Since the strands of labeled DNA and unlabeled DNA cannot be distinguished from each other, a complementary strand displacement reaction occurs. On the other hand, in No. 2, the wild type and the Δ508 mutant have a difference of 3 bases, and the complementary strand displacement reaction is difficult. For this reason, it was recognized that the fluorescence intensity of No. 2 having a mutation in the unlabeled DNA was lower than that of No. 1 having no mutation.

is Therefore, wild-type unlabeled DNA and Δ508 mutant can be obtained by adding wild-type and Δ508 mutant unlabeled DNA to wild-type labeled double-stranded DNA and performing complementary strand displacement reaction. It was confirmed that it could be distinguished from unlabeled DNA. Also, when comparing No. 3 and No. 4, the labeled double-stranded DNA

20 No. 4 in which all of the labeled DNAs are Δ508 mutant forms a complementary strand displacement reaction, and the labeled double-stranded DNA is Δ508 mutant and unlabeled DNA is wild type N It is recognized that the fluorescence intensity is higher than o.3. Therefore, by adding the wild-type and Δ508-mutated unlabeled DNA to the Δ508 mutant-labeled double-stranded DNA and performing the complementary strand displacement reaction, the wild-type unlabeled DΝ It was confirmed that 508 could be distinguished from mutant unlabeled DNA.

(Example 3)

As shown in Table 2, an experiment similar to that in Example 1 was performed by reducing the amount of labeled double-stranded DNA and the amount of unlabeled DNA to 1 Z10 equivalent. Using a fluorescence plate reader (fluorester (BMG Labtechnologies GmbH), excitation filter: 485 nm, fluorescence filter: 538 nm) for microtiter array The fluorescence intensity was measured. In addition, it measured as a control without adding unlabeled DNA. Table 2 shows the results.

Table 2

From the results in Table 2, it can be seen that the fluorescent intensities of Νο.2 and な ο.6, in which the labeled double-stranded DΝ and the unlabeled D 相補 are complementary, are higher than in the other cases. It was recognized that remarkable occurrence occurred.

In contrast, No. 3 and N◦.5, in which the labeled double-stranded D D and the unlabeled D D are different by 3 bases, have no complementary strand displacement reaction and low fluorescence intensity. It is admitted.

Therefore, it was confirmed that even if the amount of labeled double-stranded DNA and the amount of unlabeled DNA in the complementary strand displacement reaction were reduced, it was possible to distinguish between the wild type and the Δ508 mutant.

(Example 4)

A plasmid containing the nucleotide sequence of wild-type exon 10 (pCF10-3) and a plasmid containing the nucleotide sequence of the Δ508 mutant exon 10 (PCF508-1) ) Was used as a template to perform a polymerase chain reaction (PCR) using the following pair of primers (CF10 ETP, CF10 ETM).

Braima

C F 10 E T P:

SEQ ID NO: 1 in Sequence Listing

5 ― ccattaaaga aaatatcatc— ύ

C F 1 0 Ε Τ Μ ：

SEQ ID NO: 2 in Sequence Listing

o — atattcat ca taggaaacac— ύ

The PCR was performed according to a conventional method [denaturation: 30 sec at 94 C, annealing ring: 54]. A cycle of 30 sec at C and elongation: 60 sec at 72 ° C] was repeated 35 times. After the reaction, the PCR reaction solution was measured by 3% agarose gel electrophoresis. In the reaction solution from the plasmid containing the nucleotide sequence of wild-type exon 10, a band was detected at a length of 44 bases. In addition, in the reaction solution from the plasmid containing the base sequence of exon 10 of the Δ508 mutant, a band was detected at a length of 41 bases.

A complementary strand displacement reaction was performed between the PCR reaction solution prepared above and the labeled double-stranded DNAs (A) and (Β) prepared in Example 1, and a fluorescent plate reader was prepared in the same manner as in Example 3. The fluorescence intensity was measured using [Bolenostar (BMGL abtechno 1 ogies GmbH), excitation filter: 485 nm, fluorescence filter: 538 nm]. Table of results Shown in 3

Table 3

From the results in Table 3, when No. 2 and No. 3 are compared, No. 2 using wild-type labeled double-stranded DNA complementary to the wild-type PCR reaction solution causes a complementary strand displacement reaction. It can be seen that the fluorescence intensity is higher than that of No. 3 using the Δ508 mutant PCR reaction solution and wild-type labeled double-stranded DNA having three base differences.

When No. 5 and No. 6 are obtained, No. 6 using the Δ508 mutant-labeled double-stranded DNA complementary to the Δ508 mutant PCR reaction solution is a complementary strand displacement reaction. And the fluorescence intensity is higher than that of No. 5 using Δ508 mutant double-stranded DNA that differs from the wild-type PCR reaction solution by 3 bases.

Therefore, it was confirmed that it was possible to determine whether the DNA contained in the PCR reaction solution was a wild type or a Δ508 mutant type.

According to the nucleic acid identification method of the present invention, nucleic acids having a small amount of gene mutation or polymorphism in a sample can be easily and easily treated in a homogeneous system that does not require complicated solid-liquid separation. Has many mutations or polymorphisms in the sample It can simultaneously detect and quantify nucleic acids in a short time.

Further, according to the test kit of the present invention, a nucleic acid having a mutation or a polymorphism of a trace gene in a sample can be rapidly and reliably detected by a simple operation according to the identification method of the present invention. Automation is also possible, which is extremely useful in medical settings.

Therefore, according to the present invention, a small amount of mutation or polymorphism of a gene contained in a specimen can be reliably detected, and a gene showing the mutation or polymorphism can be quantified. It is extremely useful for early detection, diagnosis and treatment of cancer and specific viruses and bacterial infections in the field, and determination of the success or failure of bone marrow transplantation and the presence / absence of rejection.

Claims

The scope of the claims

1. Competitive hybridization is performed by mixing the first nucleic acid and the second nucleic acid, and the degree of substitution of the complementary strand between the two nucleic acids is measured, whereby the first nucleic acid and the second nucleic acid are mixed. In the method of identifying identity with the second nucleic acid,

At least one of two types of labels capable of energy transfer to each other is introduced into the 3 ′ end of one strand of the first nucleic acid, which is a double-stranded nucleic acid, and 5 '' The other nucleic acid is introduced into the end to label the first nucleic acid, and the labeled first nucleic acid is mixed with the unlabeled second nucleic acid, which is a single-stranded or double-stranded nucleic acid, to perform a competitive hybridization. Measuring the degree of energy change due to the energy transfer between the labels, thereby measuring the degree of displacement of the complementary strand generated between the first nucleic acid and the second nucleic acid. A method for identifying a nucleic acid, characterized by this.

2. Competitive hybridization is performed by mixing the first nucleic acid and the second nucleic acid, and the degree of substitution of the complementary strand between the two nucleic acids is measured. A method for identifying identity with a second nucleic acid, comprising:

A labeled first nucleic acid prepared by introducing at least one of two kinds of labels capable of energy transfer to each other into the first nucleic acid, and a labeled second nucleic acid prepared by introducing the other label into the second nucleic acid Are mixed in a combination of the following (a) to (c) to perform competitive hybridization, and the degree of energy change due to the energy transfer between the above-mentioned labels is measured. A method for identifying a nucleic acid, comprising measuring a degree of displacement of a complementary strand generated between a nucleic acid and a second nucleic acid.

(1) Both the first nucleic acid and the second nucleic acid are double-stranded nucleic acids, and a labeled first nucleic acid having one label introduced at the 3 ′ end of one strand, and a label introduction of the labeled first nucleic acid Combination with a labeled second nucleic acid having the other label introduced at the 5 'end of the strand to be hybridized with the strand.

(Mouth) One end of the first nucleic acid, which is a double-stranded nucleic acid, A combination of a labeled first nucleic acid into which a label has been introduced and a labeled second nucleic acid in which the other label has been introduced into the 5 ′ end of the second nucleic acid, which is a single-stranded nucleic acid.

3. The method for identifying a nucleic acid according to claim 1 or 2, wherein the at least two kinds of labels are fluorescent substances capable of energy transfer in a state of being close to each other.

4. The method for identifying a nucleic acid according to any one of claims 1 to 3, wherein the first nucleic acid and the second nucleic acid are prepared by a polymerase chain reaction (PCR).

5. The method according to claim 1, wherein one of the first nucleic acid and the second nucleic acid is a sample nucleic acid containing the target nucleic acid, and the other is a standard nucleic acid for identifying identity with the sample nucleic acid. 5. The method for identifying a nucleic acid according to any one of items 4 to 4.

6. A test kit for identifying a nucleic acid by the method according to claim 5, wherein

A sample nucleic acid amplification reagent for amplifying a specific region of the target nucleic acid in the sample to prepare a sample nucleic acid;

A standard nucleic acid amplification reagent for preparing a standard nucleic acid that identifies the identity of the target nucleic acid;

A nucleic acid inspection kit, comprising: a reagent for introducing at least two kinds of labels capable of energy transfer in a state close to each other to one or both of a sample nucleic acid and a standard nucleic acid.