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A high-throughput nucleic acid epigenetic modification quantitative analysis method

A technology of epigenetic modification and quantitative analysis, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high cost of sequencing analysis, large sample damage, etc., to reduce the amount of detection samples used and shorten the analysis time. the effect of time

Active Publication Date: 2021-12-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
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  • 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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  • A high-throughput nucleic acid epigenetic modification quantitative analysis method
  • A high-throughput nucleic acid epigenetic modification quantitative analysis method
  • A high-throughput nucleic acid epigenetic modification quantitative analysis method

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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 high-throughput nucleic acid epigenetic modification quantitative analysis method, which uses DNA polymerase to achieve quantitative analysis of methylation modification with different pause times when passing through different methylation modification sites. Specific markers on bases modified by epigenetics have large steric hindrance or contain strong polar groups to reduce the amplification efficiency and further amplify the detection signal. This method can avoid the disadvantages of cumbersome, time-consuming, and large sample usage in conventional nucleic acid methylation detection methods. Through simple and intuitive steps, it can reduce the amount of detection samples, shorten the analysis time, achieve single-base resolution, and realize DNA methylation under mild conditions. Rapid, sensitive, high-throughput quantitative detection of epigenetic modifications in the genome.

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 Patents(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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