JP2008187909A - Method for recognizing variation inside nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable recognition of variations inside nucleic acids, by using a single-stranded nucleic acid cleaving nuclease in a neutral vicinity where a double-stranded oligonucleotide has high stability. <P>SOLUTION: A mismatch sequence part of a double-stranded nucleic acid is cleaved and a variation in the nucleic acid is recognized, by making a double-stranded nucleic acid, having a mismatch sequence, react with a single-stranded nucleic acid cleaving nuclease in an alkaline buffer solution that contains a reagent to partly break a disulfide bond of the single-stranded nucleic acid cleaving nuclease. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a recognition method for discriminating SNP (Single Nucleotide Polymorphism).

  SNPs are the most common among polymorphisms present on the genome, and are estimated to exist at a rate of one place per several hundred to 1,000 base pairs. SNPs cover the entire genome compared to other DNA markers, and by comparing the SNP pattern and frequency between a group with some disease and a group of normal people, the SNP is associated with which disease. Information is obtained and has significance as a marker.

  In addition, it is well known that certain drugs have completely different effects from person to person, and one of the causes is thought to be that the amount and quality of protein expressed in the body varies slightly between individuals. It is considered that SNP is involved in this. In such a case, the SNP itself can be used for diagnosis of disease susceptibility, metabolic distribution kinetics for a drug, determination of differences in sensitivity to drugs and differences in side effects. Therefore, it is very useful to search and research SNPs.

  The most direct method for detecting SNP is to directly determine and compare the base sequences of two or more human genomes. This involves determining the base sequence randomly from small pieces of genomic DNA, computer There are a method of connecting by processing (shotgun sequence) and a method of determining a base sequence for a specific gene. In addition, DNA chips using oligonucleotide arrays (for example, Patent Document 1) and the fact that single-stranded DNA becomes double-stranded depending on the basic sequence of the DNA strand under appropriate conditions can be used for this change. SSCP method, which is detected by electrophoresis of sample with non-denaturing gel, DNA cartridge is negatively charged while column cartridge is neutral, and positively charged triethylammonium ion (TEAA) Based on the principle of binding, single-stranded DNA fragments have a reduced degree of negative charge and are faster than double-stranded fragments. DHPLC (thermal denaturing high-performance liquid chromatography) using elution from the column. ) Laws are known.

On the other hand, as a method for detecting SNP using an enzyme reaction, a method using a single-stranded nucleic acid cleavage nuclease such as S1 nuclease is known. The method using S1 nuclease utilizes the fact that S1 nuclease is an enzyme that degrades single-stranded DNA and RNA, amplifies wild type and mutant DNA of the target gene by PCR, and mismatches by hybridization This method has the advantage that it is very simple. In this method, an S1 nuclease is allowed to act on this, and the mismatched portion is cleaved and detected by electrophoresis.
JP 2005-102579 A

  However, since the optimum pH of the single-stranded nucleic acid cleaving nuclease represented by the above S1 nuclease is an acidic region around pH 4 or an alkaline region around pH 10, double-stranded DNA tends to cause dissociation. For this reason, for example, the optimum pH of S1 nuclease is about 4.5, but when SNP detection is attempted in this pH range, whether a single strand that is a mismatched portion is recognized or cleaved, Since the strand DNA becomes unstable and dissociates to become a single strand, and it is not known whether this single strand portion is recognized and cleaved, there is a problem that it is doubtful whether the SNP is correctly recognized. However, in the vicinity of neutrality where the stability of the double-stranded DNA is good, the single-stranded nucleic acid cleaving nuclease does not work, so SNP cannot be detected.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for recognizing a mutation in a nucleic acid using a single-stranded nucleic acid-cleaving nuclease near the neutrality where the stability of the double-stranded nucleic acid is good. It is what.

  The method for recognizing a mutation in a nucleic acid of the present invention comprises an alkaline buffer containing a double-stranded nucleic acid having a mismatch sequence and a single-stranded nucleic acid-cleaving nuclease containing a reagent that partially cleaves the disulfide bond of the single-stranded nucleic acid-cleaving nuclease. In which the mismatch sequence portion contained in the double-stranded nucleic acid is cleaved to recognize the mutation.

  Here, the nucleic acid is a chain in which nucleotides consisting of a base, phosphate and sugar are the basic units, and this phosphate is polymerized by forming a diester bond between the 3 ′ and 5 ′ carbons of the sugar between each nucleotide. Mean polynucleotide or oligonucleotide. RNA and DNA are divided according to whether the sugar moiety is ribose or deoxyribose.

  A double-stranded nucleic acid having a mismatch sequence is one in which the base of one nucleic acid is cytosine (C), the base of the other nucleic acid is G (guanine), and the base of one nucleic acid is adenine (A). A combination in which the base is not a complementary bond of T (thymine) (or U (uracil)), that is, a non-complementary base of CT (U), GT (U), AC, or AG And a double-stranded nucleic acid hybridized with each other in a complementary sequence portion. Even if the combination of non-complementary bases CT (U), GT (U), AC, and AG is one pair in a double-stranded nucleic acid, multiple pairs exist continuously. May be.

  The method for recognizing a mutation in a nucleic acid of the present invention is preferably subjected to electrophoresis after the cleavage. The reagent is preferably 2-mercaptoethanol.

  The single-stranded nucleic acid cleavage nuclease is preferably any one selected from the group consisting of S1 nuclease, mung bean nuclease and exonuclease I.

  The method for recognizing a mutation in a nucleic acid of the present invention comprises an alkaline buffer containing a double-stranded nucleic acid having a mismatch sequence and a single-stranded nucleic acid-cleaving nuclease containing a reagent that partially cleaves the disulfide bond of the single-stranded nucleic acid-cleaving nuclease. Because it reacts in the middle, the mismatch sequence part contained in the double-stranded nucleic acid can be specifically cleaved without causing the dissociation of the double-stranded nucleic acid, and the mutation such as SNP can be recognized accurately. It is.

  Why does a single-stranded nucleic acid-cleaving nuclease react with a double-stranded nucleic acid having a mismatch sequence in an alkaline buffer containing a reagent that partially cleaves a disulfide bond? Although it is not clear whether the mismatch sequence part contained in the double-stranded nucleic acid can be cleaved in the region, the mechanism is not clear, but the single-stranded nucleic acid cleaving nuclease is probably partially cleaved. It is presumed that the structure of the strand nucleic acid-cleaving nuclease has been changed slightly so that it works in a pH range where dissociation of double-stranded nucleic acid does not occur.

  According to the method for recognizing a mutation in a nucleic acid of the present invention, a mutation such as a gene such as SNP can be found more accurately and easily by electrophoresis or the like. Wide range of life sciences including gene expression monitoring, gene sequencing, gene polymorphism SNP analysis, gene amplification and deletion in cancer, classification of diseases such as cancer, drug responsiveness prediction, disease gene search, etc. In a DNA microarray that is expected to be applied in a target DNA, a target DNA that is not completely complementary to a probe DNA on the array but has a similar sequence will hybridize with the probe DNA on the array in error. In some cases, so-called mismatch binding may occur, and this mismatched target DNA contributes to noise generated during detection and decreases detection accuracy. However, single-stranded DNA in an alkaline buffer containing 2-mercaptoethanol is used. By reacting with a nucleic acid cleavage nuclease, the mismatch sequence contained in the double-stranded nucleic acid is cleaved. It is possible, the application of a method of removing only the polynucleotide target DNA such that the mismatch binding are also possible.

  The method for recognizing a mutation in a nucleic acid of the present invention comprises an alkaline buffer containing a double-stranded nucleic acid having a mismatch sequence and a single-stranded nucleic acid-cleaving nuclease containing a reagent that partially cleaves the disulfide bond of the single-stranded nucleic acid-cleaving nuclease. It is made to react in, the mismatch sequence part contained in a double stranded nucleic acid is cut | disconnected, and the said mutation is recognized, It is characterized by the above-mentioned.

  As an alkaline buffer, a 0.1M phosphate buffer (commercially available, or a solution prepared by mixing an appropriate amount of a sodium monohydrogen phosphate aqueous solution and a sodium dihydrogen phosphate aqueous solution to adjust the pH to 7.4), or It is preferable to use 0.1 M phosphate buffered saline (9 liters of NaCl dissolved in 1 liter of 0.1 M phosphate buffer).

  As a reagent that partially cleaves the disulfide bond of the single-stranded nucleic acid cleavage nuclease, 2-mercaptoethanol is preferable. The concentration of 2-mercaptoethanol is 0.005 to 0.02% by mass with respect to the alkaline buffer, although it depends on the temperature at which the double-stranded nucleic acid having a mismatch sequence is reacted with the single-stranded nucleic acid cleaving nuclease. It is preferable.

  The single-stranded nucleic acid cleavage nuclease is preferably any one selected from the group consisting of S1 nuclease, mung bean nuclease, and exonuclease I. These single-stranded nucleic acid cleavage nucleases can be used alone or in appropriate combination. May be used.

  Specific methods for recognizing mutations after cleaving the mismatched sequence contained in double-stranded nucleic acids are separated by electrophoresis such as electrophoresis using agarose gel or polyacrylamide gel as a carrier, or capillary electrophoresis using a capillary. Examples thereof include a recognition method and a recognition method (DHPLC method) using heat-denatured high performance liquid chromatography. Electrophoresis utilizes the fact that the migration speed varies due to differences in nucleic acid size fragments and the movement distance varies, and this method can separate nucleic acids by length and recognize mutations in the nucleic acids. Can do.

  Hereinafter, the method for recognizing a mutation in a nucleic acid of the present invention will be described in more detail with reference to examples.

(Example 1)
70mer target having the base sequence shown in FIG. 1, probe 1, probe 2, probe 3, probe 4 (the underlined portion of the base sequence of probe 2, probe 3, probe 4 corresponds to a mismatch portion with respect to the target) A nucleotide was synthesized, and a target and probe 1, a target and probe 2, a target and probe 3, and a target and probe 4 were hybridized to obtain a double-stranded oligonucleotide (duplex). FIG. 2 shows a schematic diagram of the complementarity of the probe 1, the probe 2, the probe 3, and the probe 4 with respect to the target.

  As shown in FIGS. 1 and 2, the probe 1 has a sequence that perfectly matches the target oligonucleotide (perfect match duplex control), and the probe 2 has an underlined 7mer (10% of the total sequence) at the 3 ′ end. Sequence that is mismatched to the target (3 ′ end mismatch duplex), probe 3 is a sequence in which the underlined portion 7mer at the 5 ′ end is mismatched to the target (5 ′ end mismatch duplex), and probe 4 is an underline in the sequence This is an array (intra-sequence mismatch duplex) in which the partial 7mer is mismatched with respect to the target.

  In alkaline buffer (pH 8.0) to which 0.01% by mass of 2-mercaptoethanol was added, S1 nuclease reaction (reaction temperature) was performed on 2 types of perfect match duplex and end mismatch duplex among the above 4 types of duplex. : 37 ° C. and 50 ° C., reaction time: 2 hours), followed by polyacrylamide gel electrophoresis to detect each fragment and evaluate S1 nuclease activity.

  The result of electrophoresis is shown in FIG. Each electrophoresis is a duplex (control) electrophoresis before the reaction from the left, an electrophoresis after the S1 nuclease reaction at 37 ° C., and an electrophoresis after the S1 nuclease reaction at 50 degrees. Perfect match duplex did not show S1 nuclease activity under any conditions, whereas duplex having mismatch sequences at the 3 'end and 5' end showed S1 nuclease activity at both 37 ° C and 50 ° C reaction temperatures. This showed that the S1 nuclease activity and specificity were maintained even at pH 8.

(Example 2)
For the in-sequence mismatch duplex produced above, the concentration of 2-mercaptoethanol was changed to 0.003% by mass and 0.01% by mass, and the reaction temperature was 50 ° C. in alkaline buffer (pH 8.0). After each S1 nuclease reaction for 2 hours, polyacrylamide gel electrophoresis was performed to detect each fragment, and S1 nuclease activity was evaluated.

  FIG. 4 shows the result. The electrophoresis shown in FIG. 4 has a 2-mercaptoethanol concentration of 0 (control), a 2-mercaptoethanol concentration of 0.003 mass%, and a 2-mercaptoethanol concentration of 0.01 mass% in order from the left. In the control deplex to which 2-mercaptoethanol was not added, intra-sequence mismatch duplex could not be detected. On the other hand, when the concentration of 2-mercaptoethanol was 0.003% by mass, a partial intra-sequence mismatch was partially recognized, and when the concentration of 2-mercaptoethanol was 0.01% by mass, the intra-sequence mismatch was completely recognized. From this, when 2-mercaptoethanol is not added, there is no S1 nuclease activity at pH 8, and when 2-mercaptoethanol is added, at the reaction temperature and reaction time in this example, 2- Although the concentration dependence of mercaptoethanol was recognized, it was shown that S1 nuclease activity and specificity were maintained even at pH 8.

  In this example, a combination of a plurality of non-complementary bases in a sequence was shown in a sequence, but the method for recognizing a mutation in a nucleic acid according to the present invention has one non-complementary base combination in the sequence. Even a pair can be detected. Therefore, it is also useful for detecting SNPs.

  As described above, the method for recognizing a mutation in a nucleic acid of the present invention is a method for detecting mismatches in an alkaline buffer containing a reagent that partially cleaves a disulfide bond of a single-stranded nucleic acid cleavage nuclease, such as 2-mercaptoethanol. Since the single-stranded nucleic acid and the single-stranded nucleic acid cleavage nuclease are reacted, the mismatch sequence contained in the double-stranded nucleic acid can be cleaved near the neutrality where the stability of the double-stranded nucleic acid is good. Can be recognized.

Diagram showing base sequence of synthetic oligonucleotide Schematic of probe complementarity to target Electrophoresis showing the activity of the duplex S1 nuclease Electrophoresis showing changes in S1 nuclease activity depending on 2-mercaptoethanol concentration

Claims (4)

  A method for recognizing a mutation in a nucleic acid, comprising an alkaline buffer comprising a double-stranded nucleic acid having a mismatch sequence and a single-stranded nucleic acid-cleaving nuclease containing a reagent that partially cleaves the disulfide bond of the single-stranded nucleic acid-cleaving nuclease A method for recognizing a mutation in a nucleic acid, comprising: reacting in a liquid, cleaving a mismatch sequence portion contained in the double-stranded nucleic acid, and recognizing the mutation.   The method for recognizing a mutation in a nucleic acid according to claim 1, wherein electrophoresis is performed after the cleavage.   The method for recognizing a mutation in a nucleic acid according to claim 1 or 2, wherein the reagent is 2-mercaptoethanol.   The method for recognizing a mutation in a nucleic acid according to claim 1, 2 or 3, wherein the single-stranded nucleic acid cleavage nuclease is selected from the group consisting of S1 nuclease, mung bean nuclease and exonuclease I. .