Ring mediated cascade amplification strategy used for detecting DNA transmethylase activity in high sensitivity manner

A methyltransferase and activity technology, applied in the field of enzyme activity detection, can solve the problems of non-specific background amplification, probe leakage non-specific amplification, affecting detection sensitivity and accuracy, etc. selective effect

Active Publication Date: 2017-09-12
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This partial blocking method can lead to non-specific amplification caused by probe leakage, resulting in false positive signals
In the absence of DNA methyltransferase, there will be competitive hybridization between the double-stranded probe or hairpin probe and the reporter probe, resulting in non-specific background amplification, affecting the sensitivity and accuracy of detection

Method used

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  • Ring mediated cascade amplification strategy used for detecting DNA transmethylase activity in high sensitivity manner
  • Ring mediated cascade amplification strategy used for detecting DNA transmethylase activity in high sensitivity manner
  • Ring mediated cascade amplification strategy used for detecting DNA transmethylase activity in high sensitivity manner

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Experimental program
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Effect test

Embodiment 1

[0067] 1. Analysis of Dam methyltransferase activity

[0068] To 10 μL methyltransferase buffer (50mM NaCl, 10mM Tris-HCl, 10mM MgCl 2 , 1mMdithiothreitol, pH 7.5) were added with 160μM SAM (SAM is a cofactor), 20nM long stem-loop probe, 2U DpnI and different amounts of Dam methyltransferase, and incubated at 37°C for 2h. Subsequently, 40nM hairpin probe, 1UKlenow Fragment (3′–5′exo-), 2U Nt.BbvCI and 0.6mM dNTPs and 1×CutSmart (50mM KAc, 20mM Tris-HAc, 10mM Mg( Ac) 2 , 100μg / mL BSA, pH 7.9), incubate at 37°C for 30 min. The reaction system was heated at 85 °C for 20 min to inactivate the enzyme, and then slowly cooled to 30 °C. After that, 120U T4 DNA ligase, 800nM padlock probe and 1×T4 ligase buffer (50mM Tris-HCl, 10mM MgCl 2 , 10mMdithiothreitol, 1mM ATP, pH 7.5), incubated at 37°C for 1h. Then 1mM dNTPs, 3U Phi29 DNA polymerase, 4U Nt.BbvCI and 1×CutSmart were added, and incubated at 37°C for 3h. The reaction system was heated at 75 °C for 20 min to inactivate the ...

Embodiment 2

[0074] 1. Principle analysis

[0075] The proposed rationale for the assay of methyltransferase activity is as follows: figure 1shown. When Dam methyltransferase exists, it specifically recognizes and catalyzes the methylation of long stem-loop probes to form methylated long stem-loops. Subsequently, the DpnI restriction enzyme specifically cuts the methylated long stem-loop into two parts. One part is double-stranded DNA, and the other part is new hairpin probe. Under the experimental conditions, the structure of the new hairpin probe is unstable, and a conformational transition occurs to form a single strand. Under the action of Klenow Fragment polymerase and Nt.BbvCI, the single-stranded DNA initiates polymerization and cleavage reactions, releasing multiple primers. Under the action of T4 DNA ligase, the released primer hybridizes with the padlock probe to obtain a circular probe. Subsequently, this primer triggers rolling circle amplification under the action of Phi2...

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Abstract

The invention discloses a ring mediated cascade amplification strategy used for detecting DNA transmethylase activity in a high sensitivity manner. A long-stem ring probe is designed on the basis of strand displacement amplification and exponential type rolling circle amplification. The probe comprises a methylation locus, a long-stem part and a ring part, wherein the methylation locus is used for identifying DNA transmethylase, the long-stem part is used for guaranteeing the stability of the probe, and the ring part is used for triggering subsequent amplification; the ring part and a small part of stem part are used as the triggering strands for subsequent signal output. The probe is characterized in that the triggering strands are completely sealed in the ring part of the probe by the long-stem part, and non-specific amplification caused by probe leakage is avoided; the long-stem ring probe is methylated by the DNA transmethylase and then cut by restriction endonuclease to produce the triggering strands; under the synergic effect of polymerase and the endonuclease, the produced triggering strands trigger strand displacement amplification to produce a large amount of primers; the produced primers trigger exponential type rolling circle amplification to synthesize a large amount of G-tetraploid sequences, and the sequences interact with dye to obtain enhanced fluorescent signals.

Description

technical field [0001] The invention relates to the field of enzyme activity detection, in particular to a method for highly sensitive detection of DNA methyltransferase activity using a ring-mediated cascade amplification strategy. Background technique [0002] DNA methyltransferases are epigenetic modifying enzymes that play important roles in regulating gene expression, development, and genome imprinting. It catalyzes DNA methylation by the covalent addition of methyl groups on adenine or cytosine. Studies have shown that abnormal DNA methyltransferase activity is related to the occurrence and development of diseases. Clearly, DNA methyltransferase activity is considered a potential cancer biomarker and drug target in cancer therapy. Therefore, sensitive detection of DNA methyltransferase activity is crucial for DNA methyltransferase-related cancer therapy and diagnosis. [0003] Traditional methods for the detection of DNA methyltransferase activity include radiolabel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/48C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2531/125C12Q2525/301C12Q2521/125
Inventor 姜玮王磊崔万玲
Owner SHANDONG UNIV
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