KR101136008B1 - A ligase-based SNP analysis method using oxanine base-containing ligation fragment and a system for analyzing SNP comprising the fragment - Google Patents

Abstract

The present invention comprises the steps of: a) processing a ligation fragment containing an oxanine base and a DNA ligase in a DNA template obtained from a sample comprising a target nucleic acid; And b) a single nucleotide polymorphism (SNP) analysis chip comprising a single nucleotide polymorphism (SNP) analysis method comprising analyzing the ligation reaction product of the treated product and a ligation fragment sequence containing an oxanine base, The invention relates to a particle or kit.

DNA ligase, SNP

Description

Ligase-based SNP analysis method using oxanine base-containing ligation fragment and a system using ligase-based mononucleotide polymorphism analysis method using ligation fragment sequence including oxanine base for analyzing SNP comprising the fragment}

The present invention comprises the steps of: a) processing a ligation fragment containing an oxanine base and a DNA ligase in a DNA template obtained from a sample comprising a target nucleic acid; And b) a single nucleotide polymorphism (SNP) analysis chip comprising a single nucleotide polymorphism (SNP) analysis method comprising analyzing the ligation reaction product of the treated product and a ligation fragment sequence containing an oxanine base, The invention relates to a particle or kit.

The genetic codes contained in the genomes of homologous biological entities do not coincide with each other, and differences in nucleotide sequences called polymorphisms are known. Deletion or insertion of 1 to 10 nucleotide (s), duplication of specific nucleotide sequences, and the like are known as polymorphisms. The substitution of a single nucleotide by another nucleotide is called single nucleotide polymorphisms (SNP).

Conventional methods for detecting SNPs are commonly classified as being based on hybridization, based on primer extension, and using substrate specificity of hyojo.

The presence of base substitution is detected by hybridizing the probe to the nucleic acid sample according to the hybridization method. According to this method, it is necessary to find probes and hybridization conditions so that hybridization is affected by differences on single nucleotides. Thus, it is difficult to build a highly reproducible detection system.

Mutation detection methods using cycle probe reactions (see, eg, United States Patent No. 5,660,988) illustrate conventional methods. Mutation detection methods using the taqMan method (see, eg, United States Patent Nos. 5,210,015 and 5,487,972) illustrate another method. Further examples are as follows: a method of detecting base substitution based on the presence of a primer extension reaction using the primer wherein the 3 ′ end of the primer is annealed to the nucleotide moiety for which base substitution is detected (see, eg, United States Patent No. 5,137,806; A method for detecting base substitution based on the presence of a primer extension reaction using a primer whose detected base substitution site is located from the 3 'end to the second nucleotide (see, eg, WO 01/42498); And identifying a nucleotide incorporated into the primer using a primer whose annealing of the 3 'end of the primer is located close to the 3' side relative to the nucleotide whose base substitution is detected therefor. A method of determining the presence and the nucleotides of that site. In addition, methods of using DNA ligase are known. According to this method, base substitution is detected based on the presence or absence of ligation with adjacent probes of the probe that match the detected nucleotide portion where the terminal portion of the probe is a nucleotide substitution. Additionally, methods using enzymes having the activity of recognizing and cleaving the specific structure of a double-stranded nucleic acid include, for example, invader methods (see, eg, United States Patent No. 5,846,717). One method has the following problems: The method cannot be used to detect trace target nucleic acids; Must be performed under strict temperature history conditions; Requires strict regulation to anneal the target nucleic acid; There is a need for enzymes with special properties for detection.

Subsequently, strand substitution-type nucleic acid amplification methods, such as the ICAN method (see, eg, WO 00/56877 and WO 02/16639), were developed as methods for amplifying target nucleic acids under isothermal conditions. In addition, the UCAN method (see, eg, WO 02/64833) was developed as a method of typing polymorphism of genes using DNA-RNA-DNA-type chimeric oligonucleotides.

Single nucleotide polymorphisms (SNPs) are also involved in numerous genetic diseases as changes in one DNA sequence. The need to find effective methods for detecting SNPs of human genes is rapidly increasing, which are partly compatible with the medical use of molecular diagnostic tests. Numerous SNP screening methods have already been developed. Typical examples include denaturing gradient gel electrophoresis (DGGE) assay, single-strand conformation polymorphism (SSCP) analysis, allele-specific priming of the polymerase chain reaction (allele-specific PCR), and ligase amplification reaction.

Over the past two decades, the focus of experts as a method for detecting genotype changes in unknown DNA has been focused on ligation assays (FIG. 1A). This method is very fascinating because of its specificity, speed, automation of diagnostic tests, and compatibility with PCR. Mainly thermostable DNA ligase is widely used in ligation assays. The principle of this method focuses on the fact that DNA ligase does not seal DNA sequences that contain non-complementary base pairs that do not fit together.

The traditional and most widely used DNA ligase is T4 DNA ligase. It can catalyze the ligation of pentamers faster than other known ligases. Thus, T4 DNA ligase may be an ideal candidate for fast and simple ligase-based SNP screening. However, there is a disadvantage that mismatch discrimination of other DNA ligases including T4 ligase is not high. Particularly in the case of G: T mismatch, there is an inevitable false positive result, which should not be the case for SNP discernment. (Fig. 1B).

Deng's team recently announced the use of oligonucleotide ligation assays, which are based on DNA chip methods using T4 DNA ligase (Biosens. Bioelectron, 19 (2004) 1277-1283). However, the G: T mismatch result is also shown here, and it remains the most difficult problem because the accurate C / T type analysis is difficult. Therefore, accurate detection of G: T mismatch is an obstacle and problem to be solved in T4 DNA ligase-based screening strategy for C / T type analysis of SNP.

The present invention solves the above problems, and the object of the present invention is to provide a method for detecting genotype changes of new DNA.

Another object of the present invention is to provide a system for detecting genotype changes of new DNA.

Another object of the present invention is to provide a kit for detecting genotype changes of new DNA.

In order to achieve the above object, the present invention comprises the steps of: a) processing the ligation fragment and the DNA ligase containing an oxanine base in a DNA template obtained from a sample comprising a target nucleic acid; And b) analyzing the ligation reaction product of the treated product.

As used herein, "gene polymorphism" refers to differences in the nucleotide sequence of a gene between individuals in a homogeneous community of organisms. The differences in the nucleotide sequences that make up the polymorphism are not limited to specific forms. Various types such as “base substitution”, “deletion mutations” and “insertion mutations” as described below. Differences in genetic information are also termed variations.

As used herein, “base substitution” refers to the replacement of a nucleotide with another nucleotide at a particular site of a nucleic acid. There is no particular limitation with regard to "base substitution" according to the present invention. One or more nucleotide (s) may be substituted. The substitution observed for a single nucleotide in the nucleotide sequence is named "single nucleotide polymorphism (SNP)". "Base substitution" according to the present invention also includes base substitutions artificially introduced into the nucleic acid.

The present invention is suitable for the detection of genomic polymorphisms or variations on genes, in particular base substitution (eg SNP) or insertion mutations and / or deletion mutations at gene specific sites.

Single-stranded or double-stranded nucleic acids (RNA or DNA) can be used as nucleic acids (target nucleic acids) containing genes that act as subjects for typing in the polymorphic typing method of the invention. Depending on the nuclease used, it may be difficult to use RNA as the target nucleic acid. In such cases, base substitution of RNA can be detected using the RNA as a template to prepare cDNA and using the cDNA as a target nucleic acid.

According to the invention, a sample comprising the target nucleic acid can be used for the typing reaction. Any sample that may include target nuclei such as cells, tissues (such as biopsy samples), whole blood, serum, cerebrospinal fluid, semen, saliva, sputum, urine, feces, hair, and cell culture can be used without limitation. The method of the present invention includes cell lysis and extraction and purification of nucleic acids from a sample.

In the present invention, "ligation fragment sequence containing an oxanine base" refers to a probe composed of oligonucleotides containing an oxanine base and annealed to a DNA template to participate in a ligation reaction.

In the present invention, the "upstream sequence" refers to a probe positioned on the left side of the DNA template in the 3 'end to the 5' end direction of the two probes annealed to the DNA template and participate in the ligation reaction.

In the present invention, 'downstream sequence' refers to a probe positioned to the right of a DNA template in the 3 'end to the 5' end direction among two probes annealed to the DNA template and participate in the ligation reaction.

In addition, the downstream sequence of the present invention is preferably isotopically labeled, but is not limited thereto.

The method of analyzing the ligation reaction products of fragments of the invention to determine the presence of base substitutions is determined based on the presence of the fragmented fragments after ligation. There is no particular limitation with regard to the determination method and known techniques of nucleic acid analysis can be used. Examples of methods for determining the presence of ligated fragments include: gel electrophoresis (agarose gel, polyacrylamide gel, etc.) or capillary electrophoresis to determine whether the resulting ligated product has been separated. how to check; And methods of determining whether the length of the entire fragment product has increased by ligating using mass spectrometry.

In a preferred embodiment of the present invention, the analysis method of the present invention is preferably used as a downstream sequence ligation fragment containing an oxanine base, but is not limited thereto, the DNA ligase of the present invention T 4 DNA Preferably, it is a ligase, but all enzymes with linking activity between the two fragments are included in the DNA ligase of the present invention.

In one embodiment of the present invention, the analytical method of the present invention is characterized by distinguishing G: T and G: C upstream through the results.

Further, in one embodiment, the ligation fragment sequence containing the oxanine base of the present invention is preferably the fragment described in SEQ ID NO: 4, for example, but the fragment with all the oxanine bases that can be used in the SNP analysis method is It is included in the ligation fragment of the present invention.

The nucleotide sequence of SEQ ID NO: 4 of the present invention has OCCAT TCCG ATTCT AAGTG, but there is no symbol for "O" corresponding to oxanine in the sequence cataloger. Replaced by.

When analyzing base substitutions at the genome level using the detection method of the present invention, the reaction system may be micronized to analyze a large amount of nucleotide sequences, and an integrated means for increasing the degree of integration may be used. Systems such as microchips, micro-capillary electrophoresis (CE) chips or nanochips can be used in the present invention. Accordingly, the present invention provides a chip for SNP analysis in which a ligation fragment containing an oxanine sequence is immobilized.

The present invention also provides particles for SNP analysis in which ligation fragments containing an oxanine sequence are immobilized.

A method of preparing a chip or particle by immobilizing nucleic acid on a solid surface as in the present invention is described in detail in US Pat. No. 5,215,882, etc. In summary, the method of immobilization comprises a modified nucleic acid strand and an anchor comprising a variable portion. Reacting a moiety, wherein the various moieties have a selected base sequence and anchor moiety comprising a modified at least one nucleotide base sequence having a primary amine function or an equivalent nucleotide moiety having a primary amine function in the presence of a reducing agent The free aldehyde group on the solid surface reacts with the modified nucleic acid strand to form a complex of the modified nucleic acid strand with at least a portion of the free aldehyde group on the solid surface.

In another aspect, the present invention provides a kit for SNP analysis, containing the ligation fragment sequence containing an oxanine base and DNA ligase as an active ingredient.

The present invention includes the above-mentioned gene polymorphism assay kit for ligation fragments that can be used in the method of the present invention. Although not particularly limited with respect to the fragments, for example, fragments having SEQ ID NO: 4 are preferred. Nucleotide sets comprising one of four types of nucleotides, each of which can be used simultaneously to determine whether a base substitution is present and the type of substituted nucleotides.

In addition, kits of the present invention may include DNA ligase suitable for ligation, buffers suitable for the reaction, and the like. The kit may also include reagents for detecting ligation products.

Kits of the invention may comprise probes or primers for detecting internal standards. In addition, the kit may comprise nucleic acid as an internal reference added to a test sample.

Hereinafter, the present invention will be described in more detail.

In the present invention, in the ligation of the upstream sequence and the downstream sequence, the form in which the 5 'terminal downstream sequence was replaced with Oxa, that is, the O: C form, was treated with T4 DNA ligase. If you use regular Watson and Crick pairing without oxa base pairing, you will get a false positive result regardless of whether the 3 'terminal upstream matches. The C / T type of the SNP site cannot be distinguished (FIG. 1B). On the other hand, ligation does not occur when the SNP site is G: T base pairing when using oxanine (FIG. 2B).

In order for C / T type analysis using oxanine to be reliable, bases of template sequences paired with oxanine must also be considered. Any base of C, T, G, or A can be located. Therefore, we conducted the ligation experiment in four cases: O: C, O: T, O: G, or O: A. As a result of FIG. 2C, it can be seen that ligation occurs in G: C but not G: T. When the oxa-containing DNA sequence is used as a ligation fragment, successful C / T type analysis is possible by enabling specific ligation only when the preceding sequence is G: C regardless of base pairing of oxanine.

As can be seen from the above, when an oxanine base such as Oxa-containing DNA sequence is used as a ligation fragment, it can be specifically ligated only when the preceding sequence is G: C regardless of base pairing of oxanine. Successful C / T type analysis is possible so that fragments containing damaged bases can be used for new ligase-based SNP analysis.

In order to solve the problems described above, the inventors have developed a way of using damaged nucleotides. A fragment sequence comprising the damaged form of guanine, oxanine (Oxa, O; Figure 2A left) base, is provided as a substrate to T4 DNA ligase. As a result, it was shown that T4 DNA ligase was sensitive to the match of the 3 'end upstream sequence or the 5' end downstream sequence. We hypothesized that the damaged base of the 5 'end downstream sequence will affect the linkage of the SNP site (3' end upsteam). And whether the success of the ligation will tell what the type of SNP.

First, the inventors found that two strands of 20-mer DNA ligation sequence (5'-N: 5'-CTCAG GTCGA CAGTC TGCG N-3 '(SEQ ID NO: 1), N-3': 5'-NCCAT TCCG ATTCT AAGTG- 3 '(SEQ ID NO: 2), N = G, A, T, C, O) and template strand 40mer sequence (3'N _a N _b -5': 3'-GAGTC CAGCT GTCAG ACGC N _a N _b GGTA AGGAC TAAGA TTCAC-5 '(SEQ ID NO: 3), N _a N _b = CC, TC, CT, TT, CG, TG, CA, TA) was prepared through solid-phase synthesis and RP-HPLC purification. Oxa containing ligation sequences (5′-O and O-3 ′) were also prepared by the above method.

Next, the 3 'terminal downstream sequence was labeled to confirm the ligation. The downstream sequence (800 nM) was in 50 μl reaction solution (50 mM Tris-HCl [pH 8.0], 10 mM MgCl ₂ , 5 mM DTT) with T4 polynucleotid kinase (40 unit), [r- ³² P] ATP (4.5 MBq). It reacted at 37 degreeC for 30 minutes. The reaction was terminated by heating at 85 ° C. for 10 minutes and purified by CENTRI-SEP column.

Finally, 60 μl reaction solution (50 mM Tris-HCl) with 20-mer upstream sequence (600 nM), isotopically labeled downstream (600 nM) and 40-mer template DNA sequence (600 nM) with T4 DNA ligase (5 unit) (pH 7.5), 10 mM MgCl ₂ , 10 mM DTT, 1 mM ATP, 25 μg / ml BSA). Likewise, the reaction was terminated by heating at 85 ° C. for 10 minutes, and the reaction was separated by 6M urea 20% denaturing PAGE. PAGE results were observed using a STORM 820 phosphor-imaging scanner.

1A is a diagram illustrating a DNA ligase-based SNP detection system. If it is not known whether the type of SNP is 3'-CN1-5 '(type C) or 3'-TN1-5' (type T), it can be determined by a ligation method. The SNP of the target sequence is of type C if ligated when using the upstream with the 3 ′ end of G.

1B is a diagram showing the problem of detection of G: T mismatch in T4 ligase-based SNP analysis. In the conventional method of ligation, a false positive response occurs even though the underlined T and G mismatch each other. In this case, SNP type analysis is difficult.

2 (A) shows the damaged base, (B) shows the efficiency of the Oxa-containing fragments, and (C) shows the ligation effect of normal nucleobase and oxanine base pairs.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> A ligase-based SNP analysis method using a damaged or modified          base-containing ligation fragment and a system for analyzing SNP          configuring the fragment <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide <400> 1 ctcaggtcga cagtctgcgn 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide <400> 2 nccattccga ttctaagtg 19 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide <400> 3 gagtccagct gtcagacgcn nggtaaggac taagattcac 40 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide <400> 4 nccattccga ttctaagtg 19  

Claims (20)

a) treating the DNA template obtained from the sample containing the target nucleic acid with the ligation fragment containing the oxanine base and the DNA ligase; And b) single nucleotide polymorphism (SNP) analysis method comprising analyzing the ligation reaction product of the treatment product. The SNP analysis method according to claim 1, wherein the method uses a ligation fragment containing an oxanine base as a downstream sequence. The method of claim 2, wherein the downstream sequence is isotopically labeled. According to claim 1, wherein the DNA ligase SNP analysis method, characterized in that the T4 DNA ligase. delete The SNP analysis method according to claim 1 or 2, wherein the analysis method distinguishes G: T and G: C upstream from the results. The SNP analysis method according to claim 1, wherein the ligation fragment sequence is a fragment set forth in SEQ ID NO: 4. The method of claim 1, wherein the ligation fragment sequence containing the oxanine base is immobilized on a chip or particle. SNP analysis chip in which a ligation fragment containing an oxanine sequence is immobilized. delete The SNP analysis chip of claim 9, wherein the ligation fragment sequence is a fragment described in SEQ ID NO: 4. 11. The SNP analysis chip of claim 9, wherein the ligation fragment is an isotopically labeled fragment. Particles for SNP analysis in which ligation fragments containing oxanine sequences are immobilized. delete The particle of claim 13, wherein the ligation fragment sequence is a fragment set forth in SEQ ID NO: 4. 15. The particle of claim 13, wherein the ligation fragment is an isotopically labeled fragment. SNP analysis kit containing the ligation fragment sequence containing an oxanine base and DNA ligase as an active ingredient. delete The kit according to claim 17, wherein the ligation fragment sequence is a fragment set forth in SEQ ID NO: 4. 18. The kit for analyzing SNP of claim 17, wherein the DNA ligase is T4 DNA ligase.

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