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Method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification

A technology of methyltransferase and strand displacement reaction, which is applied in the field of enzyme detection, can solve the problem of low sensitivity, and achieve the effect of wide application, good selectivity and specificity

Active Publication Date: 2015-12-02
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

developed a simple fluorescent method based on hairpin fluorescent DNA probes, however, due to the target-to-signal ratio of 1:1, the sensitivity of this method is relatively low, and the detection limit is 0.8 U / mL

Method used

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  • Method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification
  • Method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification
  • Method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification

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Embodiment 1

[0041] M.SssI, HaeIII, HhaI and AluI methyltransferase, HpaII restriction endonuclease, KlenowFragment (3′-5′exo-) polymerase, Nb.BbvCI cleavage enzyme and S-adenosylmethionine from NEB Purchased. 5-azacytidine and 5-aza-2'-deoxycytidine were obtained from Sigma-Aldrich. Four kinds of deoxyribonucleic acid (dNTPs) and HEPES were purchased from Shanghai Shenggong Biotechnology Co., Ltd. The chemicals used in the experiment are all analytically pure, and no further purification is required during use. All solutions are prepared with ultrapure water (> 18.25MΩ·cm). The oligonucleotides in this work were synthesized and purified by Shanghai Shenggong Biotechnology Co., Ltd., and their sequences are listed in Table 1.

[0042] The fluorescence spectra of all samples were measured on the Hitachi F-7000 fluorescence spectrophotometer. The emission spectrum ranges from 505 to 600 nm, and the excitation wavelength is 494 nm. The fluorescence intensity at 518nm was used to evaluate th...

Embodiment 2

[0055] Principle analysis

[0056] The principle of the proposed methyltransferase activity analysis is as follows figure 1 Shown. We have designed a trifunctional dsDNA probe, including methylation sites for DNA methyltransferase recognition, 8-17 DNAzyme complementary sequences for DNAzyme synthesis, and cleavage sites for cleavage by cleavage enzymes. First, M.SssI methyltransferase specifically recognizes and catalyzes the methylation of the trifunctional dsDNA probe to form methylated dsDNA. Subsequently, the HpaII restriction endonuclease specifically cleaves the remaining unmethylated dsDNA. Subsequently, under the action of KlenowFragment polymerase and dNTPs, P1 in the methylated dsDNA is used as a primer to initiate a polymerization reaction to form a complete DNA double strand with a cutting site. Next, Nb.BbvCI cleaves the entire DNA double-strand to create a new replication site for the next stage of polymerization. Through this repeated polymerization and cleavag...

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Abstract

The invention relates to a method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification. A three-function double-stranded DNA probe is designed. Methylation happening to the three-function double-stranded DNA probe is specifically recognized through DAN methyltransferase. Remaining non-methylated double-stranded DNA is specifically cut through HpaII restriction enzymes, methylated double-stranded DNA triggers a strand displacement reaction, a large amount of 8-17 DNAzyme is released, the 8-17 DNAzyme catalyzes cutting of a large number of hairpin-line molecular beacon substrates, and remarkable fluorescent enhancement is triggered. The method can sensitively detect the DAN methyltransferase activity, and the detection limit is 0.0082 U / mL. The method has potential application in research of influences of anti-cancer drugs on DAN methyltransferase activity inhibition and screening of DAN methyltransferase inhibitors.

Description

Technical field [0001] The invention relates to the field of enzyme detection, in particular to a method for detecting DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification. Background technique [0002] DNA methyltransferase catalyzes DNA methylation and plays a key role in regulating gene expression and development. Abnormal DNA methyltransferase activity leads to abnormal DNA methylation, which is closely related to many types of diseases such as cancer. Obviously, DNA methyltransferase activity is considered as a potential cancer biomarker and target of drug action in cancer therapy. Therefore, the development of a sensitive and selective method for the analysis of DNA methyltransferase activity and the screening of its inhibitors (anti-methylation drugs) is essential for early cancer diagnosis and treatment. [0003] Traditional methods for DNA methyltransferase activity detection include gel electrophoresis, high performance liq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/48
CPCC12Q1/48C12Q1/6818C12Q1/6844G01N2333/91017C12Q2521/125C12Q2521/301C12Q2525/301C12Q2563/107C12Q2565/1015
Inventor 姜玮王磊崔万玲
Owner SHANDONG UNIV
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