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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

Inactive Publication Date: 2014-08-20
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, none of these methods propose simultaneous confirmation of methylation-specific sites and detection of methyltransferase activity

Method used

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

Examples

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Embodiment 1

[0039] An analysis method based on restriction endonuclease and exonuclease III label-free fluorescent detection of DNA methylation, the detection steps are:

[0040] Hybridization step: in 50mM Tris-HCl (100mM NaCl, 5mM MgCl 2 , 0.1mg / mL BSA, 1mM DTT pH7.9) into the reaction system, add 5μL of 10μM probe DNA and 5μL of 10μM target DNA, mix thoroughly, react in a constant temperature water bath at 70°C for 10 minutes, cool down to room temperature naturally, and then Add TO, and the final concentration of thiazole orange in the buffer is 3.0 μM. After reacting at room temperature for one hour, the product solution is tested for fluorescence, and this solution is used as a control solution.

[0041] Methylation detection steps: add 7U methyltransferase and 50μM SAM to the DNA hybridization solution and mix thoroughly, react in a constant temperature water bath at 37°C for 1 hour, then add 10U HpaⅡ and 25U ExoⅢ, mix well, continue to After reacting in a constant temperature wat...

Embodiment 2

[0044] An analysis method based on restriction endonuclease and exonuclease III label-free fluorescent detection of DNA methylation, the detection steps are:

[0045] Hybridization step: in 10mM Tris-HCl (300mM NaCl, 2mM MgCl 2 , 0.05mg / mL BSA, 0.05mMDTT pH7) into the reaction system, add the final concentration of 20nM probe DNA and 20nM target DNA, mix thoroughly, react in a constant temperature water bath at 70°C for 10 minutes, and then cool down to room temperature naturally. Then add TO, the final concentration of thiazole orange in the buffer is 0.7 μM, after reacting at room temperature for one hour, the product solution is tested for fluorescence, and this solution is used as the control solution.

[0046] Methylation detection steps: add methyltransferase to the DNA hybridization solution with a final concentration of 1U / mL and 50μMSAM, mix thoroughly, and react in a constant temperature water bath at 37°C for 1 hour, then add HpaⅡ and ExoⅢ, the final concentration o...

Embodiment 3

[0049] An analysis method based on restriction endonuclease and exonuclease III label-free fluorescent detection of DNA methylation, the detection steps are:

[0050] Hybridization step: in 80mM Tris-HCl (500mM NaCl, 10mM MgCl 2 , 5mg / mL BSA, 10mM DTTpH7.5) into the reaction system, add probe DNA and 50nM target DNA at a final concentration of 50nM, mix well, react in a constant temperature water bath at 70°C for 10 minutes, and cool down to room temperature naturally. Then, TO was added, and the final concentration of thiazole orange in the buffer was 1.6 μM. After reacting at room temperature for one hour, the product solution was tested for fluorescence, and this solution was used as a control solution.

[0051] Methylation detection step: add the methyltransferase in the DNA hybridization solution to a final concentration of 10U / mL and 50μM SAM in the buffer, mix well, react in a constant temperature water bath at 37°C for 1 hour, and then add HpaⅡ and ExoⅢ, the final con...

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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.

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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Application Information

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IPC IPC(8): C12Q1/68C12Q1/48C12Q1/34
CPCC12Q1/683C12Q2521/125C12Q2521/301C12Q2521/319
Inventor 卫伟高春燕刘松琴
Owner SOUTHEAST UNIV
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