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Method for positioning and quantitatively detecting 6-methylaminopurine in DNA and RNA

A methylaminopurine, quantitative detection technology, applied in the fields of molecular biology and nucleic acid chemistry, can solve the problems of high cost, inability to locate methylation sites, low efficiency, etc., and achieve the effect of short pretreatment time

Inactive Publication Date: 2014-09-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is expensive, cumbersome, and inefficient, and it cannot realize large-scale genetic testing and exact methylation site positioning, which has great limitations.

Method used

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  • Method for positioning and quantitatively detecting 6-methylaminopurine in DNA and RNA
  • Method for positioning and quantitatively detecting 6-methylaminopurine in DNA and RNA
  • Method for positioning and quantitatively detecting 6-methylaminopurine in DNA and RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Design of the system to be tested

[0031] (1) Design two types of DNA template strands, one of which is ordinary DNA with adenine (DNA-A), its sequence is 5'-CCCACCCTATAGAGTCGTA-3'; the other is modified with 6-methylaminopurine DNA (DNA-m 6 A), whose sequence is 5'-CCCm 6 ACCCTATAGAGTCGTA-3', the primer strand is 5'-GTGCATGTGGCAG-3'.

[0032] (2) Design two types of RNA template strands, one of which is an ordinary RNA with adenine (RNA-A), and its sequence is 5'-CCCACCCUAUAGAGUCGUA-3'; the other is an RNA with N 6 -Methyladenine-modified RNA (RNA-m 6 A), whose sequence is 5'-CCCm 6 ACCCUAUAGAGUCGUA-3', the primer strand is 5'-GTGCATGTGGCAG-3'.

[0033] (3) Prepare 10 groups containing different concentrations of RNA-A and RNA-m 6 A sample of mixed template strands, where RNA-A and RNA-m 6 The concentration ratio of A is 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 0:10 in sequence.

Embodiment 2

[0034] Example 2: Performing a DNA replication reaction

[0035] (1) Prepare a buffer solution containing 10 mM tris hydrochloride, 50 mM ammonium sulfate, 10 mM potassium chloride, 100 mM magnesium sulfate and 100 mM polyethylene glycol octylphenyl ether; Add the following substances to the buffer solution in μL to make it final concentration: Bst 0.5 U for polymerase, 400 μM for 5-hydroxymethyldeoxyuridine triphosphate (5-hmdUTP), and 200 nM for primer strand;

[0036] (2) Add the designed two types of DNA template strands respectively, the total concentration is 300 nM, at 50 o Incubate in a water bath of C for 60 min;

[0037] (3) Add 500 μL quencher (volume fraction of formamide is 80%, disodium edetate concentration is 200 mM, pH=8.0) to the reaction system, 80 o C for 20 min, then naturally cooled to room temperature;

[0038] (4) The reaction solution was subjected to polyacrylamide gel electrophoresis (denaturing polyacrylamide gel with a volume fraction of 12%) ...

Embodiment 3

[0039] Embodiment 3: Carry out RNA reverse transcription reaction

[0040] (1) Prepare a buffer solution containing 2 mM tris hydrochloride, 10 mM ammonium sulfate, 1 mM potassium chloride, 20 mM magnesium sulfate and 20 mM polyethylene glycol octylphenyl ether; Add the following substances to the buffer solution in μL to make it final concentration: Bst 0.2 U for polymerase, 0.2 U for RNase inhibitor, 100 μM for 5-fluorouracil deoxynucleotide (5-F-dUTP), and 200 nM for primer strand;

[0041] (2) Add the two types of RNA template strands designed respectively, the total concentration is 500 nM, at 70 o Incubate in a water bath of C for 20 min;

[0042] (3) Add 500 μL quencher (volume fraction of formamide is 80%, disodium edetate concentration is 200 mM, pH=8.0) into the reaction system, 100 o C for 10 min, then naturally cooled to room temperature;

[0043] (4) Electrophoresis the reaction solution on polyacrylamide gel (denaturing polyacrylamide gel with a volume fract...

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Abstract

The invention discloses a method for positioning and quantitatively detecting 6-methylaminopurine in DNA and RNA. The methylation level of nucleic acid is reflected by detecting 6-methylaminopurine in DNA and RNA, wherein substrate activity in DNA replication and RNA reverse transcription will be reduced due to the existence of 6-methylaminopurine, and in the DNA replication and RNA reverse transcription process, chain extension will not occur when methylation loca occur; therefore, deoxyribonucleoside triphosphate can be doped in for chain extension so as to differentiate the methylation sequence and the non-methylation sequence, and meanwhile the methylation level of a specific locus of DNA and RNA can be quantitatively evaluated. The method can be well used for analyzing 6-methylaminopurine in nucleotide separation of DNA and RNA and can be used for detecting the methylation level of adenine in DNA and RNA easily and conveniently.

Description

technical field [0001] The invention belongs to the fields of molecular biology and nucleic acid chemistry, and relates to a method for positionally and quantitatively detecting 6-methylaminopurine in DNA and RNA. Background technique [0002] Epigenetics is a subject gradually developed in the process of studying life phenomena that do not conform to the classic Mendelian inheritance rules, also known as "pseudogenetics", "epigenetics", "exogenous" or " Post-genetics", which mainly studies reversible, heritable changes in function without changes in the deoxyribonucleic acid (DNA) in the nucleus. Methylation refers to the process of catalytically transferring methyl groups from active methyl compounds (such as S-adenosylmethionine) to other compounds, forming various methyl compounds, or for certain proteins or nucleic acids etc. for chemical modification to form methylated products. DNA methylation is a mechanism of epigenetics. It adds a methyl group to a DNA molecule, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2565/125
Inventor 周翔王少儒田沺王佳琪翁小成
Owner WUHAN UNIV
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