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A high-throughput online detection method for the dynamic process of DNA methylation

A detection method and dynamic process technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., can solve the problem that there is no effective method for dynamic DNA methylation process, so as to reduce non-specific interference, The effect of short analysis time and low measurement cost

Active Publication Date: 2020-08-04
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no effective method for online detection of dynamic DNA methylation process

Method used

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  • A high-throughput online detection method for the dynamic process of DNA methylation
  • A high-throughput online detection method for the dynamic process of DNA methylation
  • A high-throughput online detection method for the dynamic process of DNA methylation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Effect of DNA methylation on FRET effect

[0059] Using DNA methyltransferase DNMT3a to act on the 5'-C methylation site CG Take G-3' as an example.

[0060] (1) Prepare 500 µL sample stock solution (S 1 ), which contains 500nM labeled 5-FAM DNA probe strand, 500nM labeled 5-FAM complementary DNA strand and 0.1M phosphate buffer (pH=7.4).

[0061] (2) Prepare 500 µL sample stock solution (S 2 ), which contains 500nM labeled 5-FAM methylated DNA probe strand (fluorescent label at the 3' end), 500nM labeled 5-FAM methylated DNA complementary strand (fluorescent label at the 3' end of the methylation site nucleotides within 3bp in the terminal direction) and 0.1M phosphate buffer (pH=7.4). S 2 In addition to being methylated, the DNA sequence is identical to S 1 The DNA sequences in are identical.

[0062] (3) Extract 50µL of S 1 , S 2 To the cuvette, detect the fluorescence signal in the fluorescence instrument (excitation light EX: 488nm, emission lig...

Embodiment 2

[0066] Example 2 Action state of methyltransferase at different temperatures

[0067] Using DNA methyltransferase DNMT3a to act on the 5'-C methylation site CG Take G-3' as an example.

[0068] (1) Prepare 1mL sample stock solution (S 3 ), which contains 500nM labeled 5-FAM DNA hybrid strand (wherein the fluorescent label of one DNA single strand is on the nucleotide within 3bp from the methylation site to the 3' end, and the fluorescent label of the other complementary sequence is on 3' end, both fluorescent labels are 5-FAM), 1×NE buffer (pH=7.4), 150nM DNMT3a, 150µM SAM; the 1×NE buffer contains 50 mM NaCl (sodium chloride) , 10 mM Tris-HCl (tris(hydroxymethyl)aminomethane-hydrochloric acid solution), 10 mM MgCl 2 (magnesium chloride) and 1mM DTT (dithiothreitol). .

[0069] (2) Extract 50µL of S 3 to the cuvette, detect the fluorescence signal (EX: 488nm, EM: 500~640nm) in the fluorometer, and record the initial signal value ( F S3-0 ).

[0070] (3) will S 3 Pla...

Embodiment 3

[0074] Example 3 Optimization of methyltransferase adsorption time

[0075] (1) Prepare 4 parts of 1mL sample stock solution (S 4 , S 5 , S 6 , S 7 ), each DNA hybrid strand containing 500nM labeled 5-FAM (the fluorescent label of one DNA single strand is on the nucleotide within 3bp of the methylation site to the 3' end, and the fluorescent label of the other complementary sequence At the 3' end, both fluorescent labels are 5-FAM), 1×NE buffer (pH=7.4), 150nM DNMT3a, 150µM SAM. The 1×NE buffer contains 50 mM NaCl (sodium chloride), 10 mM Tris-HCl (tris(hydroxymethyl)aminomethane-hydrochloric acid solution), 10 mM MgCl 2 (magnesium chloride) and 1mM DTT (dithiothreitol).

[0076] (2) Extract 50µL of S 4 , S 5 , S 6 , S 7 to the cuvette, detect the fluorescence signal in the fluorometer (EX: 488nm, EM: 500~640nm), record the initial fluorescence signal value ( F S4-0 , F S5-0 , F S6-0 , F S7-0 ).

[0077] (3) S 4 , S 5 , S 6 , S 7 Place it in a constant te...

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Abstract

The invention discloses a high-throughput online detection method for the dynamic methylation process of DNA (deoxyribonucleic acid). The method includes: using a PCR (polymerase chain reaction) instrument to detect a mixture of fluorescence-labeled target DNA double strands, transmethylase, transmethylase substrate and a buffer solution through a detection procedure at 36-38 DEG C for 6-8 min and another procedure at 48-50 DEG C for 2.5-3.5 min; performing at least 10 cycles; recording fluorescence values in the detection process. The nondestructive detection method for the methylation process of DNA is established herein, any reaction other than methylation is involved, and the detection results are closest to the true state of a subject; the method is universal to studies on methylation of specific sites; a detection method of secondary labeling or secondary reaction is not required; the method of the invention has the advantages of simpler operational steps, short analytical time, and low measurement cost.

Description

technical field [0001] The invention relates to a high-throughput online detection method for the dynamic process of DNA methylation. Background technique [0002] DNA methylation refers to the transfer of a methyl group to the C5 position of cytosine by catalyzing the substrate S-adenosylmethionine (SAM) under the action of methyltransferases (such as DNMT3a) to generate 5-methyl Cytosine (5-mC) process. As one of the important epigenetic modification methods, DNA methylation is closely related to genetic inheritance and embryonic development, and is also a potential marker of diseases such as tumors. The mode and degree of DNA methylation modification are closely related to the mode of action and activity of MTases. The study of DNA methylation mechanism and analysis method is of great significance for in-depth understanding of epigenetic mechanism, cell growth process and diagnosis of major diseases. [0003] DNA methylation analysis methods are divided into genome-wid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12Q1/48
CPCC12Q1/48C12Q1/6858G01N2333/91011C12Q2531/113C12Q2521/125C12Q2563/107
Inventor 戴宗张立邹小勇
Owner SUN YAT SEN UNIV
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