Method for detecting activity of DNA methylase and DNA methyltranseferase by unlabeled fluorescent detection based on restriction endonuclease and exonuclease III

A technology of restriction endonuclease and exonuclease, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problem of not proposing and confirming methyltransferase at the same time, so as to avoid the high cost of detection Effect
CN103993083AInactive Publication Date: 2014-08-20SOUTHEAST UNIV
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
SOUTHEAST UNIV
Publication Date
2014-08-20
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses a method for detecting the activity of DNA methylase and DNA methyltranseferase based on restriction endonuclease Hpa pi and exonuclease III. An unlabeled DNA probe is prepared and the sequence of the probe comprises a cleavage site of the restriction endonuclease and a methylated CpG site. The purpose of detecting the activity of the methylase and methyltranseferase is reached by steps of hybridizing the unlabeled DNA probe with the target DNA, carrying out methylation treatment by the methylase, carrying out the specific cleavage of the restriction endonuclease Hpa pi and enabling exonuclease III having the activity of 3'->5' exonuclease to act on the double-stranded DNA and then adding fluorescence signal molecules thiazole orange which having different signals to single-stranded DNA and double-stranded DNA. According to the method, since no complex material or labeling of DNA probes need to be prepared, the defects of high detection cost, cumbersome operations and poor reproducibility caused by the preparation of the material and labeling of DNA probes are avoided. The method disclosed by the invention has the advantages of low cost and high sensitivity and is fast and simple.
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Description

technical field

[0001] The invention belongs to the field of biological detection, and specifically relates to a detection method for unlabeled fluorescent detection of DNA methylation and methyltransferase activity by restriction endonuclease and exonuclease III. The invention belongs to a method for identifying a special methylation site Biosensing technology for point and quantitative analysis of DNA methyltransferase activity, specifically refers to DNA methylation, after being recognized by methylation-sensitive restriction endonucleases and cutting specific sequences, exonuclease III digestion DNA, to observe the change of the fluorescent signal molecule thiazole orange signal. Background technique

[0002] DNA methylation is an important modification pathway. Under the catalysis of methyltransferase, the cytosine of the CG two nucleotides of DNA is selectively added with a methyl group to form 5-methylcytosine, which is common in the 5'-CG-3' sequence of the gene (kn...

Claims

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