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Method for identifying and detecting 5-hydroxymethyl cytosine and 5-formyl cytosine in DNA

A technology of hydroxymethylcytosine and formylcytosine, which is applied in the field of identification and detection of 5-hydroxymethylcytosine and 5-formylcytosine in genomic DNA, can solve the problem of cumbersome steps, expensive equipment, and damage to biological tissues environment and other problems, to achieve the effect of simple steps and high sensitivity

Inactive Publication Date: 2019-06-21
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods are able to achieve the detection of 5-hydroxymethylcytosine, they still have some limitations
For example, thin-layer chromatography will cause different degrees of damage to biological tissues and cause environmental pollution due to the use of radioactive isotopes; other methods have cumbersome steps or expensive equipment

Method used

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  • Method for identifying and detecting 5-hydroxymethyl cytosine and 5-formyl cytosine in DNA
  • Method for identifying and detecting 5-hydroxymethyl cytosine and 5-formyl cytosine in DNA
  • Method for identifying and detecting 5-hydroxymethyl cytosine and 5-formyl cytosine in DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0044] This example is the detection of sequences containing cytosine, 5-methylcytosine and 5-hydroxymethylcytosine at different positions.

[0045] 1. The oligonucleotide HEX-31mer-hmC17 (X=5hmC) whose sequence is 5'-TAATTGCGGTCAATTG(X)GGATCATGCGTATA-3' is used as a template for detection, and cytosine or 5-methylcytosine exists at the X site Other DNA sequences of pyrimidines (HEX-31mer-C17, X=C; HEX-31mer-mC17, X=5mC) were used as controls.

[0046] 2. Label the 5' end of the DNA sequence with 5'-hexachlorofluorescein-CE phosphoramidite (HEX). Add 200 nM of DNA to a solution containing 50 mM NaOH and 60 μM KRuO 4 The mixed solution was reacted in an ice-water bath for 60 min, the oxidized DNA sequence was purified by a small rapid spin nucleotide purification column, and the obtained DNA sequence was placed in 300 mM NaOH aqueous solution at 95 ° C for 15 min. After cooling to 4°C, a deionized formamide solution with a mass fraction of 80% was added to terminate the react...

Embodiment 2

[0049] Exploring the Oxidation Reaction KRuO 4 The effect of concentration and treatment time on the cleavage efficiency of sequences containing 5-hydroxymethylcytosine sites (HEX-31mer-hmC17 containing a single 5-hydroxymethylcytosine site). Oxidation reaction in the presence of 50mM NaOH and different concentrations of KRuO 4 The mixed solution was carried out in an ice-water bath. Handle different times. The product DNA was purified by filtration through a semi-permeable membrane, and 3M NaOH stock solution was added to ensure the final alkali concentration was 300mM, and incubated at 95°C for 15min.

[0050] Results: When the oxidation reaction was carried out at 200 μM KRuO 4 Carry out 40min in middle and then carry out thermo-alkali reaction, cracking efficiency is the highest ( image 3 ). Higher concentration of KRuO 4 or longer reaction times will result in fewer cleavage products.

Embodiment 3

[0052] This embodiment is used for the detection of sequences with multiple 5-hydroxymethylcytosine sites.

[0053] 1. The oligonucleotide HEX-31mer-hmC17 (X=C, Y=5hmC, Z=C), HEX- 31mer-hmC17-hmC26 (X=C, Y=5hmC, Z=5hmC) and HEX-31mer-hmC7-hmC17-hmC26 (X=5hmC, Y=5hmC, Z=5hmC) were used as templates.

[0054] 2. Mark the 5' ends of the three DNA sequences with HEX, and use the method in step 2 of Example 1 to process the DNA sequences.

[0055] Results: It was observed that HEX-31mer-hmC17 containing only one 5-hydroxymethylcytosine site had only one cleavage fragment ( Figure 4 band 2), and the correct length after cleavage of HEX-31mer-hmC17-hmC26 containing two 5-hydroxymethylcytosine sites can be observed in the results of denaturing polyacrylamide gel analysis ( Figure 4 Strip 4). Not approved by KRuO 4 Oxidized DNA remained intact under the same alkaline conditions and no cleavage was observed in denaturing polyacrylamide gel analysis ( Figure 4 Strips 1, 3 and 5)....

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Abstract

The invention discloses a method for identifying and detecting 5-hydroxymethyl cytosine (5hmC) and 5-formyl cytosine (5fC) in DNA and belongs to the fields of molecular biology, nucleic acid chemistryand epigenetics, The method includes: adding to-be-detected DNA into a mixed solution containing potassium perruthenate and alkali, performing reaction under an ice water bath condition to allow the5hmC in the DNA to be oxidized into the 5fC; purifying the DNA after the reaction, adding into a hot alkali solution to perform heat-preservation reaction to achieve DNA cleavage, and performing gel analysis to measure the total number and site of the 5hmC and 5fC in the DNA; directly adding the to-be-detected DNA into the hot alkali solution without potassium perruthenate oxidation to perform heat-preservation reaction to achieve DNA cleavage, and performing gel analysis to measure the total number and site of the 5fC in the DNA; comparing the two results to obtain the number and site of the5hmC and 5fC in the DNA. By the method high in sensitivity and wide in application range, the defects that existing detection methods are high in equipment requirements, complex in operation and the like.

Description

technical field [0001] The invention belongs to the fields of molecular biology, nucleic acid chemistry and epigenetics, and specifically relates to a method for identifying and detecting 5-hydroxymethylcytosine and 5-formylcytosine in genomic DNA. 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] DNA methylation is the most important modification method in epigenetics. It refers to S-adenosyl methionine (S-adenosyl methioni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6809
Inventor 赵轩王少儒刘奕侬万泽中王天洋李惠蒋尚文范若晨张楠
Owner WUHAN UNIV
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