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A method for detecting the methylation modification at N6 or N1 position of adenine in nucleic acid by using dutp or dttp

A methyladenine and nucleic acid technology, applied in the field of identification and detection of N6-methyladenine or N1-methyladenine in the genome, to achieve the effect of mild experimental conditions

Inactive Publication Date: 2021-08-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the previously developed methods also have certain problems.

Method used

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  • A method for detecting the methylation modification at N6 or N1 position of adenine in nucleic acid by using dutp or dttp
  • A method for detecting the methylation modification at N6 or N1 position of adenine in nucleic acid by using dutp or dttp
  • A method for detecting the methylation modification at N6 or N1 position of adenine in nucleic acid by using dutp or dttp

Examples

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Effect test

Embodiment 1

[0049] The method of the invention is applied to sequence detection under 25 μM dUTP. The specific steps are as follows:

[0050] 1.6-carboxyfluorescein (6-carboxy-fluorescein, FAM) has a maximum excitation wavelength of 495nm and a maximum absorption wavelength of 521nm, which can be used to label the 5' end of the DNA primer to be tested. Under the excitation of ultraviolet light in a gel imager, No re-staining is required for imaging;

[0051] 2. Prepare 1 μL of 1×ThermoPol buffer at pH 8.8 at 25° C. according to the following formula: 20 mM Tris-HCl, 10 mM ammonium sulfate, 10 mM potassium chloride, and 2 mM magnesium sulfate. Add 0.1% polyethylene glycol octylphenyl ether, 0.2 μL double-stranded DNA with a final concentration of 2 μM (containing adenine at the same position, N 6 -Methyladenine, N 1-methyladenine) DNA-17mer-A1, DNA-17mer-6mA1 and DNA-17mer-1mA1, 25 μM dUTP, 1 μL Bst DNA polymerase, and primer Primer, the concentration ratio of primer and double-stranded...

Embodiment 2

[0056] The method of the present invention is applied to sequence detection under 6.25 μM dTTP, and the specific steps are as follows:

[0057] 1. Prepare 1 μL of 1×ThermoPol buffer at pH 8.8 at 25°C: 20 mM Tris-HCl, 10 mM ammonium sulfate, 10 mM potassium chloride, and 2 mM magnesium sulfate. Add 0.1% polyethylene glycol octylphenyl ether, 0.2 μL double-stranded DNA with a final concentration of 2 μM (containing adenine at the same position, N 6 -Methyladenine, N 1 -Methyladenine) DNA-17mer-A1, DNA-17mer-6mA1 and DNA-17mer-1mA1, 6.25 μM dTTP, 1 μL Bst DNA polymerase, and primer Primer, the concentration ratio of primer and double-stranded DNA is 3:5 , and adding ddH 2 O make up the volume to 10 μL;

[0058] 2. Incubate at a controlled constant temperature of 63° C. for 5 minutes, take samples for analysis at different intervals, and add 45 μL of stop buffer (95% formamide, 25 mM EDTA, pH 8.0) to stop the reaction during analysis. Immediately after termination, heat to 90°...

Embodiment 3

[0062] The method of the present invention is used to explore whether spermine exists to contain N 1 -The detection impact of the sequence of the methyladenine site, the specific steps are as follows:

[0063] 1. Prepare two groups of 1 μL 1×ThermoPol buffer solution at pH 8.8 at 25° C. according to the same recipe as in Examples 1 and 2. Add 0.1% polyethylene glycol octylphenyl ether, 0.2 μL double-stranded DNA DNA-17mer-1mA1 with a final concentration of 2 μM, dTTP with a final concentration of 6.25 μM, 1 μL Bst DNA polymerase, and primer Primer, primer and double-stranded The concentration ratio of DNA was 3:5, one group was added with a final concentration of 100 μM spermine, the other group was used as a blank control, and ddH was added to both systems 2 O make up the volume to 10 μL.

[0064] 2. Incubate at a controlled constant temperature of 63° C. for 10 min, and add 45 μL of stop buffer (95% formamide, 25 mM EDTA, pH 8.0) to terminate the reaction. Immediately aft...

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Abstract

The invention discloses a method for detecting the methylation modification at the N6 or N1 position of adenine in nucleic acid by using dUTP or dTTP. The detection method is mainly composed of two parts: the first part is to incorporate deoxyribonucleotide uridine triphosphate (dUTP) or deoxyribonucleotide triphosphate (dTTP) into the DNA sequence through an extension reaction. The second part is to obtain the extension percentage of chains containing different sites through denaturing polyacrylamide gel analysis. After processing, the extension rate of chains containing different sites can be judged to achieve identification and detection. 6 ‑Methyladenine and N 1 ‑Methyladenine purpose. The method overcomes the shortcomings of existing detection methods such as high equipment requirements, expensive raw materials, cumbersome operations, etc., and has high sensitivity and wide application range.

Description

technical field [0001] The invention belongs to the field of molecular biology, nucleic acid chemistry and epigenetics (Epigenetic), in particular to a kind of N 6 -Methyladenine (N 6 -methyladenine) or N 1 - A method for the identification and detection of methyladenine. Background technique [0002] As an important branch of genetics, epigenetics mainly studies the heritable changes in gene expression based on non-gene sequence changes. Its mechanism of action mainly includes: DNA methylation, chromatin remodeling, non-coding RNA regulation, histone modification, etc. Epigenetics is related to many life functions, such as genome imprinting, X-chromosome inactivation, etc. At the same time, studies have shown that epigenetics is important for the occurrence of diseases such as cancer, immune system diseases, and several inherited mental retardation diseases. [0003] In epigenetics, the modification of RNA is very rich, and there are about hundreds of kinds. Studies h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6809
CPCC12Q1/6809C12Q2537/164C12Q2521/107
Inventor 赵轩田沺王少儒宋燕燕王天洋蒋尚文万泽中李惠范若晨张楠
Owner WUHAN UNIV
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