A method for the quantitative detection of 6-methylaminopurine in dna and rna

A technology for quantitative detection of methylaminopurine, applied in the fields of molecular biology and nucleic acid chemistry, can solve the problems of expensive, cumbersome, and inability to locate methylation sites, and achieve the effect of short pretreatment time

Inactive Publication Date: 2016-06-08
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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  • A method for the quantitative detection of 6-methylaminopurine in dna and rna
  • A method for the quantitative detection of 6-methylaminopurine in dna and rna
  • A method for the quantitative detection of 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 a common DNA with adenine (DNA-A) whose 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 a common adenine-bearing RNA (RNA-A) whose sequence is 5'-CCCACCCUAUAGAGUCGUA-3'; 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 RNA-A and RNA-m with different concentrations 6 A sample of mixed template strands with RNA-A and RNA-m 6 The concentration ratios of A were 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 0:10.

Embodiment 2

[0034] Example 2: Performing DNA Replication Reactions

[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 octyl phenyl ether; add 20 μL of the buffer solution The following substances make their final concentrations: Bst 0.5U of polymerase, 400 μM of 5-hydroxymethyldeoxyuridine triphosphate (5-hmdUTP), and 200 nM of primer strand;

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

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

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

Embodiment 3

[0039] Example 3: Performing 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 octyl phenyl ether; add 20 μL of the buffer solution The following substances make their final concentrations: Bst 0.2 U of polymerase, 0.2 U of RNase inhibitor, 100 μM of 5-fluorouracil deoxynucleotide (5-F-dUTP), and 200 nM of primer strand;

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

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

[0043] (4) The reaction solution was electrophoresed on a polyacrylamide gel (denaturing polyacrylamide ge...

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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 positioning and quantitatively detecting 6-methylaminopurine in DNA and RNA. Background technique [0002] Epigenetics is a discipline gradually developed in the process of studying life phenomena that do not conform to the classical Mendelian laws of inheritance, also known as "pseudogenetics", "epigenetics", "epigenetics" or " "Postgenetics", which studies reversible, heritable changes to function in the absence of changes in deoxyribonucleic acid (DNA) in the nucleus. Methylation refers to the process of catalyzing the transfer of methyl groups from active methyl compounds (such as S-adenosylmethionine) to other compounds, which can form various methyl compounds, or can be used for certain proteins or nucleic acids. chemical modification to form methylated products. DNA methylation is a mechanism of epigenetics. It adds methyl groups to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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