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Quantitative analysis method for epigenetic modification of high-throughput nucleic acid

A technology for epigenetic modification and quantitative analysis, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high cost of sequencing analysis, large sample damage, etc., to reduce the amount of test samples used and the time used to be short. , the effect of shortening the analysis time

Active Publication Date: 2019-01-11
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the bisulfite reaction is very damaging to the sample, and the cost of sequencing analysis is very high

Method used

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  • Quantitative analysis method for epigenetic modification of high-throughput nucleic acid
  • Quantitative analysis method for epigenetic modification of high-throughput nucleic acid
  • Quantitative analysis method for epigenetic modification of high-throughput nucleic acid

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Experimental program
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Effect test

Embodiment 1

[0084] The establishment of embodiment 1 analytical method

[0085] Take 5'-GAG ACC GGA GTC CGC TTT CCT CTT CCG GAA AAT GTA AGC CGA ACC TAAAGC AAT CAC CAG GG-3' (SEQ ID NO.1) as an example.

[0086] (1) Preparation of nucleic acid fragments with different epigenetic modifications

[0087] The 8 DNA template sequences are as follows:

[0088] C: 5'-GAG ACC GGA GTC CGC TTT CCT CTT CCG GAA AAT GTA AGC CGA ACC TAAAGC AAT CAC CAG GG-3' (SEQ ID NO.1);

[0089] 5mC: 5'-GAG ACC GGA GTC CGC TTT CCT CTT CCG GAA AAT GTA AGC mCGA ACCTAA AGC AAT CAC CAG GG-3' (SEQ ID NO.2);

[0090] 5hmC: 5'-GAG ACC GGA GTC CGC TTT CCT CTT CCG GAA AAT GTA AGC hmCGA ACCTAA AGC AAT CAC CAG GG-3' (SEQ ID NO.3);

[0091] 5fC: 5'-GAG ACC GGA GTC CGC TTT CCT CTT CCG GAA AAT GTA AGC fCGA ACCTAA AGC AAT CAC CAG GG-3' (SEQ ID NO.4);

[0092] 5caC: 5'-GAG ACC GGA GTC CGC TTT CCT CTT CCG GAA AAT GTA AGC caCGA ACCTAA AGC AAT CAC CAG GG-3' (SEQ ID NO.5);

[0093] A: 5'-GGA CTG GAC TGG ACT GGA CTG GAC TAT CAC CAG G...

Embodiment 2

[0135] Application of embodiment 2 analytical method

[0136] (1) Optimization of reaction conditions for isothermal amplification of total DNA from actual samples

[0137] Optimization of conditions for total DNA methylation assay in real samples by single factor rotation method

[0138] (a) template usage

[0139] Fix the amount of primer (100nM), prepare DNA fragment solutions with different concentrations, and optimize the optimal template amount. The reaction was prepared in two parts, Part A and Part B. Part A included nicking enzyme buffer (1×), dNTPs (250 μM), primer strands and template solutions of different concentrations, and Part B included polymerase buffer (1× ), SYBR Green II fluorescent dye (2×), KF (exo-) polymerase, Nt.BsmAI Nease nickase, all the above operations were performed on ice. Part A and Part B were mixed, and quickly placed in a quantitative PCR instrument for amplification reaction at 37°C. The reaction concentrations of DNA were 81.75, 65.40...

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Abstract

The invention discloses a quantitative analysis method for epigenetic modification of a high-throughput nucleic acid. The quantitative analysis of methylated modification is realized by utilizing thedifference of pause time when a DNA (deoxyribonucleic acid) polymerase passes different methylated modification object loca; an epigenetic modified basic group to be tested is specifically marked witha group which has relatively large steric hindrance or relatively strong polarity to reduce the amplification efficiency and further amplify a detection signal. The quantitative analysis method can avoid the shortcomings such as complication, time consumption, large dosage of samples and the like in a conventional nucleic acid methylation detection method; through simple and intuitive steps, thedosage of the detection sample is reduced, and the analysis time is shortened; the resolution ratio of the single basis group is achieved; and the rapid, sensitive, high-throughput quantitative detection of epigenetic modification in a genome under mild conditions is realized.

Description

technical field [0001] The invention relates to a high-throughput nucleic acid epigenetic modification quantitative analysis method. Background technique [0002] The 5th carbon atom of cytosine (C) on the CpG dinucleotide site in DNA is methylated and modified under the catalysis of DNA methyltransferase, and the process of generating 5-methylcytosine (5mC) is important One of the methods of epigenetic modification of nucleic acid. This methylation modification is a reversible process. Under the action of TET enzyme, 5mC will be gradually oxidized to generate other epigenetic modification methods, including 5-hydroxycytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), and through base The excision repair (BER) process returns cytosines. DNA methylation modification almost runs through the entire genome and participates in many physiological processes, such as gene expression, transcription, gene imprinting, embryonic development, chromosome structure, et...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2531/119
Inventor 戴宗陈丹萍邹小勇
Owner SUN YAT SEN UNIV
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