KR20140000734A - Highly-efficient ligase-based snp analysis using dna ligation fragments comprising an non-binding modified nucleobase - Google Patents
Highly-efficient ligase-based snp analysis using dna ligation fragments comprising an non-binding modified nucleobase Download PDFInfo
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- KR20140000734A KR20140000734A KR1020120067434A KR20120067434A KR20140000734A KR 20140000734 A KR20140000734 A KR 20140000734A KR 1020120067434 A KR1020120067434 A KR 1020120067434A KR 20120067434 A KR20120067434 A KR 20120067434A KR 20140000734 A KR20140000734 A KR 20140000734A
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Abstract
The present invention relates to a high efficiency ligase-based SNP analysis using a DNA ligation fragment containing a non-binding modified base, and more particularly, to a non-binding modified base of a downstream ligation fragment in a ligase-based SNP analysis. Successful C / T type by introducing at the 5 'end and using as a substrate for ligase, allowing specific ligation only when the preceding sequence is G: C match regardless of base pairing of non-binding modified base Polymorphic analysis is possible.
Description
The present invention relates to a DNA ligation fragment comprising a non-binding modified base applicable to the field of ligase-based SNP analysis.
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).
Single nucleotide polymorphisms (SNPs) are involved in numerous genotypes as a single DNA nucleotide sequence change. SNP is distributed at one probability per approximately 1000 sequences and is a major cause of the difference in the incidence of genetic diseases and drug resistance among individuals. SNP has four types (C: G-> T: A, C: G-> G: C, C: A-> G: T, depending on the change of four bases (A, G, C, T)). T: A-> A: T), of which about 70% of C / T type SNPs are polymorphisms between cytosine (C) and thymine (T).
As the importance of SNP analysis increases due to individual disease prediction and personalized medicine, efficient SNP detection methods have been continuously studied, and numerous SNP screening methods have already been developed. Representative examples include single-strand conformation polymorphism (SSCP) analysis and mini-sequencing method of allele-non-specific method, RT-PCR method and ligase assay using molecular beacon of allele-specific method. have.
Over the past two decades, numerous SNP screening methods have been developed, but ligase-based SNP assays have been suggested as a viable method in terms of assay efficiency (FIG. 1A). This method has several advantages. First of all, multiplex analysis is possible, and it is possible to combine SNP analysis of various samples at the same time by combining with micro analysis methods such as flow cytometry, micro array, micro fluidics, and capillary. In addition, fusion can be easily applied to nanoparticles such as gold nanoparticles, which are methods for enhancing signal amplification and specificity, or optical imaging using FRET. In particular, when ligation analysis is linked with PCR, a high signal can be obtained even with a small concentration of sample, and integration with existing DNA-based automation systems is possible. However, T4 DNA ligase and thermal DNA ligase have a fundamental problem in obtaining a ligation result that distinguishes a G: C match and a G: T mismatch at the terminal portion of the ligation junction. In other words, the method of distinguishing SNPs based on the success of ligation is fast and simple, so that fusion application with various methods is possible, but there is a false positive result in which a reaction occurs in a G: T mismatch, especially among ligase reactions. There is this. In other words, DNA ligase is inevitably positive even in the non-complementary binding of G: T mismatch, and as a result of this inaccurate result, DNA ligase lowers the reliability of the system for screening C / T type SNPs using ligase. This is a large factor (Fig. 1C). Deng's team published a DNA chip-based ligation-based SNP analysis using T4 DNA ligase, which also revealed false positive errors in which G: T mismatch was ligated. Identifying C / T type differences by identifying G: C matches and G: T mismatches as successes and failures of the ligation reactions, respectively, is a challenge in developing a ligase-based SNP analysis system.
It is an object of the present invention to provide a new SNP analysis technique for detecting genotype changes in DNA.
In order to achieve the above object, the present invention provides a probe for ligase-based single nucleotide polymorphism (SNP) analysis comprising a downstream ligation fragment containing a non-modified modified nucleobase.
The present invention also provides a chip for SNP analysis in which a ligase-based SNP analysis probe according to the present invention is immobilized.
The present invention also provides particles for SNP analysis in which the ligase-based SNP analysis probe according to the present invention is immobilized.
The present invention also provides a kit for SNP analysis comprising a probe for ligase-based SNP analysis according to the present invention.
The present invention also comprises the steps of reacting a nucleic acid template for polymorphism analysis, a ligase-based SNP analysis probe and a ligase according to the present invention; And
It provides a SNP analysis method comprising the step of analyzing the ligation reaction product.
In the present invention, when the non-binding modified base is bound to the end of the downstream ligation fragment and used for ligase-based SNP analysis, the present invention is only used when the preceding sequence is G: C regardless of the base pairing of the non-binding modified base. By enabling specific ligation, successful C / T type polymorphism analysis is possible.
1 illustrates the principle of C / T type SNP analysis using ligase. A) is an ideal case where no ligation product is observed during mismatch (G: T) assuming that ligase has high substrate specificity. B) shows the undermatched T and G mismatched state when the L4 is detected as a general substrate during G: T mismatch detection in T4 DNA ligase-based SNP analysis using the technique of A). Even though it shows a result of false positive reaction, C) when the non-binding modified base is introduced at the 5 'end of the downstream ligation fragment according to the present invention. It is possible to detect C / T type SNP without false positive in the ligation reaction.
Figure 2A is a type (A) of the non-binding modified base introduced at the 5 'end of the downstream ligation fragment according to the present invention, and cytosine (C) by using a downstream ligation fragment containing the non-binding modified base C / T type SNP analysis results (B) performed on existing nucleic acid templates and C / Ts performed on nucleic acid templates containing other bases, such as thymine (T), adenine (A), and guanine (G). Type SNP analysis result (C) is shown.
EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.
In ligase-based SNP analysis, the present inventors considered a method in which a non-binding modified base is introduced at the 5'-end of a downstream ligation fragment and used as a substrate of ligase. In general, the ligase-based SNP analysis method is to detect the difference in ligation response according to the upstream 3'-end match / mismatch. In the present invention, the 5'-end of the downstream ligation fragment is used in such a conventional system. By additionally removing the complementary binding of the junction, we tried to solve the false positive problem that occurred. This complementary non-binding was solved by placing the non-binding modified base at the 5'-end of the downstream ligation fragment, which introduced the base without the complementary binding at the ligation junction site on the template and the upstream ligation fragment. The success or failure of the ligation is determined according to whether the 3'-end of the match / mismatch.
Accordingly, the present invention relates to probes for ligase-based monobasic polymorphism (SNP) analysis comprising downstream ligation fragments containing a modified nucleobase.
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" are 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.
Probes of the invention are suitable for detecting genomic polymorphisms or variations on genes, in particular base substitution (eg SNP) or insertion mutations and / or deletion mutations at gene specific sites.
The probe of the present invention refers to a probe composed of oligonucleotides containing a non-binding modified base and annealed to a nucleic acid template to participate in a ligation reaction.
In the present invention, the term "upstream ligation fragment" refers to a probe positioned on the left side of the nucleic acid template in the 3'-end to the 5'-end direction of two probes annealed to the nucleic acid template to participate in the ligation reaction. it means. The upstream ligation fragment can be coupled to a detectable substance to detect the ligation product. Examples of detectable materials include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; Examples of suitable prosthetic molecule complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, monosyl chloride or phycoerythrin ); Examples of luminescent materials include luminol; Examples of bioluminescent materials include luciferase, luciferin and acuorin; Suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H.
In the present invention, a "downstream ligation fragment" refers to a probe positioned to the right of a nucleic acid template in the 3- 'to 5'-end direction of two probes annealed to the nucleic acid template to participate in the ligation reaction. Means. The downstream ligation fragment of the present invention may comprise the sequence set forth in SEQ ID NO: 1, but any downstream ligation fragment that can be used for SNP analysis can be used without limitation.
In the present invention, "non-binding modified base" refers to a modified base that does not have complementary binding to a base while leaving the molecular binding capacity of the ligase, and may be located at the 5 'end of the downstream ligation fragment, for example 7-deaza-2'deoxyinosine, 2'-OMe inosine, in addition to nitroindole, spacer, dSpacer, Iso-dG and Iso-dC -OMe inosine, 2'-OMe 3-nitropyrrole, 4-nitrobenzimidazole, 4-aminobenzimidazole, nubula Nebularine, 2'O-methoxyethyl inosine, 2'-O-methoxyethyl nebularine, 2'-O-methoxy Indole such as ethyl 4-nitro-benzimidazole, 2'-O-methoxyethyl 3-nitropyrrole, Imidazole, or pyrrole derivatives may be used.
The probe of the present invention may be used as a substrate of T4 DNA ligase or thermal DNA ligase, but is not particularly limited thereto.
The probes of the present invention can be used to distinguish G: T mismatches and G: C matches at the ends of upstream ligation fragments, but are known according to the type of non-binding modified base that has no complementary binding at the ligation junction site on the template. It can be used to distinguish different types of polymorphism.
When analyzing base substitution at the genome level using the probe 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 the ligase-based SNP analysis probe is fixed.
The present invention also provides particles for SNP analysis in which the ligase-based SNP analysis probe is immobilized.
The chips or particles of the present invention are prepared by immobilizing the probe of the present invention on a solid surface, and the method of preparation is described in detail in US Pat. Reacting the nucleic acid strand with the anchor moiety, wherein the various moieties have a selected base sequence and anchor moiety comprising a modified at least one nucleotide base having primary amine function or an equivalent nucleotide base having primary amine function and a reducing agent In the presence, 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.
The present invention also relates to an SNP assay kit comprising a ligase-based SNP assay probe according to the present invention.
The SNP assay kit of the invention comprises a downstream ligation fragment of the invention. Although not particularly limited with respect to the fragment, it may include, for example, the sequence set forth in SEQ ID NO: 1. It can be used to include a set of nucleotides, each containing one of four types of nucleotides, which can be used to simultaneously determine the presence of a base substitution and the type of substituted nucleotide.
In addition, the kits of the present invention may include DNA ligase suitable for ligation, buffers suitable for 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.
The present invention also comprises the steps of reacting a nucleic acid template for polymorphism analysis, a ligase-based SNP analysis probe and a ligase according to the present invention; And
It relates to a SNP analysis method comprising the step of analyzing the ligation reaction product.
As the nucleic acid template for the polymorphism analysis, a nucleic acid (target nucleic acid) containing a single- or double-stranded nucleic acid (RNA or DNA) acting as an analysis target in the SNP analysis method of the present invention can be used.
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 in the assay. Any sample that can include target nucleic acids such as cells, tissues (such as biopsy samples), whole blood, serum, cerebrospinal fluid, semen, saliva, sputum, urine, feces, hair and cell cultures can be used without limitation. The method of the present invention includes cell lysis and extraction and purification of nucleic acids from a sample.
Ligation reaction of the nucleic acid template, probe and ligase using a DNA ligase of 40U to 200U, can be carried out at 10 to 20 ℃, more specifically at about 16 ℃ at 2 to 12 hours, nucleic acid template Depending on the reaction conditions may vary, it is not particularly limited thereto.
The method of analyzing the ligation reaction product of fragments of the present invention to determine the presence of base substitution is determined based on the presence of the ligation 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 ligating using mass spectrometry to determine whether the length of the entire fragment product has increased.
In a preferred embodiment of the present invention, the assay method of the present invention may utilize a ligation fragment containing a non-binding modified base such as 5-nitroindole, dSpacer, Iso-dG, Iso-dC, etc. as a downstream sequence. As the ligase, T4 DNA ligase or thermal DNA ligase may be used, but any enzyme having a linking activity between two fragments may be used without limitation.
In one embodiment of the invention, the analytical method of the present invention is characterized by distinguishing the G: T mismatch from the end of the upstream ligation fragment and the G: C match.
Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
Example 1 Probe Preparation and SNP Analysis for Ligase-Based SNP Analysis
Each sequence of the ligation fragment was synthesized by chemical method:
Upstream Ligation Fragment (20mer, 5'-G): 5 '-/ FAM / CTCAG GTCGA CAGTC TGCG)
Downstream ligation fragments (20mer, N m -3 '): / N m / CCATTCCTGATTCTAAGTG-3', N m : 5-Nitroindole, dSpacer, Iso-dG and Iso-dC)
Template (40mer, 3'-N a N b -5 '): 3'-GAGTCCAGCTGTCAGACGC / N a N b / GGTAAGGACTAAGATTCAC-5', N a N b = CC, TC, CT, TT, CG, TG, CA, TA
The ligation result was fluorescently labeled at the 5'-end of the upstream ligation fragment for confirmation, and a phosphate group was further introduced at the 5'-end of the downstream ligation fragment for the ligase reaction.
Next, ligation analysis was performed using T4 DNA ligase, and the conditions were as follows.
The upstream ligation fragment (1 μM), the downstream ligation fragment (1 μM) and the template (1 μM) were reacted with T4 DNA ligase (100-200 U) in 25 μl reaction solution for 16 ° C. for 12 hours. In order to terminate the reaction and denature the sample, 5 μl of loading buffer (TaKaRa, 10 ×) and 11 μl of urea solution (12M) were added together and heated at 90 ° C. for 5 minutes, and then rapidly cooled to 4 ° C. and cooling. ). The reaction was separated on 7M urea, 12% denaturation PAGE and analyzed by image analyzer (ImageQuant ™ LAS 4000).
As shown in Figures 1C and 2, accurate C / T type SNP analysis using T4 DNA ligase was obtained by applying downstream ligation fragments with non-binding modified bases introduced at the 5'-end.
In general, the 3'-end of an upstream ligation fragment should be classified as a match / mismatch success or failure. As described above, G: T has a false positive result which is a problem even for a mismatch. 2B, lanes 2 and 7). Here, when a non-binding modified base such as Nitroindole (NI), dSpacer (AP), Iso-dG, Iso-dC is introduced at the 5'-end of the downstream ligation fragment, the 3'-end of the upstream ligation fragment In the G: C match (type C), the ligation proceeds successfully (Fig. 2B, lanes 3-6), but in the case of G: T mismatches, no false-positive results are found. 2B, lanes 8-11).
The experiment was performed by analyzing the template strand (3'-C C -5 '/ 3'-T C -5') having cytosine (C) at a complementary position to the modified base. In this regard, it was examined whether the same effect is applied to other bases (thymine (T), adenine (A), guanine (G)) other than cytosine (C).
As shown in Figure 2C, each modified base, AP, 5-NI. Probes containing IsodG and IsodC do not have false positives when mismatching, regardless of all nucleotide types in the complementary position of the template strand, whereas high levels of false positives were found in the control group using normal probes. It became.
The ligation process described above is controlled in the range of T4 DNA ligase (40U ~ 200U) and reaction time (2 ~ 12 hours at 16 ℃) within a certain range for optimum efficiency when applied to various samples (template) in the field It can be seen that it is necessary.
The present invention uses downstream ligation fragments introduced with non-binding modified bases to clearly distinguish the success of ligation results according to the 3'-end match / mismatch of the upstream ligation fragment.
In the non-binding modified bases exemplified in the present invention, almost all of the results of false-positive in G: T mismatch (T type) ligation were hardly detected. Among the modified bases used, C-T type SNP classification efficiency was the highest when 5-nitroindole was used.
Successful C / T type SNP analysis based on the ligase of the present invention utilizes a ligated fragment incorporating a modified base having a non-base-binding modified base at the end of the ligase without any molecular binding ability. It is shown that accurate ligase-based SNP analysis that does not occur is possible, and in particular, accurate C / T type SNP analysis results are presented.
<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Highly-efficient ligase-based SNP analysis using DNA ligation fragments comprising an non-specific binding modified nucleobase <130> P120438 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Downstream ligation fragment <400> 1 ccattcctga ttctaagtg 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Upstream ligation fragment <400> 2 ctcaggtcga cagtctgcg 19 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Template: NN = CC, TC, CT, TT, CG, TG, CA, TA <400> 3 gagtccagct gtcagacgcn nggtaaggac taagattcac 40
Claims (12)
Non-binding modified bases include 7-deaza-2'deoxyinosine, 2'-OMe, in addition to nitroindole, spacer, dso-dG, and Iso-dC. 2'-OMe inosine, 2'-OMe 3-nitropyrrole, 4-nitrobenzimidazole, 4-aminobenzimidazole ), Nebularine, 2'O-methoxyethyl inosine, 2'-O-methoxyethyl nebularine, 2'- 2'-O-methoxyethyl 4-nitro-benzimidazole and 2'-O-methoxyethyl 3-nitropyrrole One or more ligase-based monobasic polymorphism (SNP) analysis probes selected from the group consisting of:
A non-binding modified base is a ligase-based SNP analysis probe included in the 5 'end of the downstream ligation fragment.
The downstream ligation fragment is a ligase-based SNP analysis probe comprising the nucleotide sequence of SEQ ID NO: 1.
Ligase is a ligase-based SNP analysis probe that is T4 DNA ligase or thermal DNA ligase.
A probe for ligase-based SNP analysis, wherein the probe distinguishes a G: T mismatch from a upstream ligation fragment end and a G: C match.
SNP analysis method comprising the step of analyzing the ligation reaction product.
Ligase is T4 DNA ligase or thermal DNA ligase (thermal DNA ligase) SNP analysis method.
Ligation reaction product analysis is to distinguish between G: T mismatch and G: C match at the end of the upstream ligation fragment.
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| WO2021045591A1 (en) * | 2019-09-05 | 2021-03-11 | 고려대학교 세종산학협력단 | Oxanine nucleobase-mediated dna ligation method |
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