A method for detecting dna methyltransferase activity based on strand displacement amplification and dnazyme amplification

A technology of methyltransferase and strand displacement reaction, applied in the field of enzyme detection, can solve problems such as low sensitivity, and achieve the effects of wide application, good selectivity and specificity

Active Publication Date: 2019-03-05
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, with a detection limit of 0.8 U / mL

Method used

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  • A method for detecting dna methyltransferase activity based on strand displacement amplification and dnazyme amplification
  • A method for detecting dna methyltransferase activity based on strand displacement amplification and dnazyme amplification
  • A method for detecting dna methyltransferase activity based on strand displacement amplification and dnazyme amplification

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Experimental program
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Embodiment 1

[0041] M.SssI, HaeIII, HhaI and AluI methyltransferases, HpaII restriction endonuclease, Klenow Fragment (3′–5′exo-) polymerase, Nb.BbvCI cutter and S-adenosylmethionine from Purchased from NEB. 5-azacytidine and 5-aza-2'-deoxycytidine were obtained from Sigma-Aldrich. Four deoxyribonucleic acid (dNTPs) and HEPES were purchased from Shanghai Sangon Biotechnology Co., Ltd. All chemicals used in the experiments were of analytical grade and used without further purification. All solutions were prepared with ultrapure water (>18.25MΩ·cm). The oligonucleotides in this work were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd., and their sequences are listed in Table 1.

[0042] The fluorescence spectra of all samples were measured on a 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 518 nm was used to evaluate the performance of this sensing...

Embodiment 2

[0055] Principle analysis

[0056] The proposed rationale for the assay of methyltransferase activity is as follows: figure 1 shown. We designed a trifunctional dsDNA probe, including a methylation site for DNA methyltransferase recognition, an 8–17 DNAzyme complementary sequence for DNAzyme synthesis, and a cleavage site for nickase cleavage. First, M.SssI methyltransferase specifically recognizes and catalyzes the methylation of the trifunctional dsDNA probe to form methylated dsDNA. Subsequently, the HpaII restriction enzyme specifically cleaves residual unmethylated dsDNA. Subsequently, under the action of Klenow Fragment polymerase and dNTPs, P1 in the methylated dsDNA acts as a primer to initiate the polymerization reaction, forming a complete DNA double strand with a cleavage site. Next, Nb.BbvCI cleaves the complete DNA duplex, creating a new replication site for the next stage of polymerization. Through this repeated polymerization and cleavage reaction, a large n...

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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 methyltransferases catalyze DNA methylation and play key roles 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. Clearly, DNA methyltransferase activity is considered a potential cancer biomarker and drug target 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 (antimethylation drugs) is crucial for early cancer diagnosis and treatment. [0003] Traditional methods for the detection of DNA methyltransferase activity include gel electrophoresis, high performance liquid ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12Q1/6818C12Q1/48
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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