Kit and method for detecting activity of DNA methyltransferase

A kit and methylation technology, applied in the field of biological analysis, can solve the problems of low sensitivity, simple instrument, non-specific exponential amplification, etc., and achieve the effect of high sensitivity

Inactive Publication Date: 2018-12-18
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Typical examples include fluorometric assays based on exonuclease III-assisted target recycling and endonuclease (e.g., Nt. / mL, 0.01U / mL and 0.04U / mL; and chemiluminescence analysis based on rolling circle amplification (RCA), which is simple in instrument but not high in sensitivity, and the detection limit is 1.29×10 -4 U / mL, in addition, both involve the careful design of molecular beacon probes and the preparation of complex circular templates, and are also subject to high background signals caused by non-specific digestion or non-specific amplification; it is worth noting that conventional nucleic acid Amplification techniques such as polymerase chain reaction (PCR), strand displacement amplification (SDA), rolling circle amplification (RCA), and isothermal exponential amplification (EXPAR) reactions are usually based on DNA polymerase or DNA polymerase The combination of enzymes and nicking enzymes to generate large amounts of DNA fragments for signal amplification, but they all inevitably suffer from high background signal caused by non-specific amplification, the reason can be attributed to: (1) some DNA aggregation The enzyme does not correct the exonuclease activity to repair the mismatched deoxyribonucleotides, which will lead to non-specific fragments; (2) DNA polymerase-mediated de novo synthesis and polymeric extension of DNA double strands, making the endonucleic acid Enzyme recognition sites are incorporated randomly, which leads to non-specific exponential amplification

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  • Kit and method for detecting activity of DNA methyltransferase
  • Kit and method for detecting activity of DNA methyltransferase
  • Kit and method for detecting activity of DNA methyltransferase

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Experimental program
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Effect test

Embodiment 1

[0083] Circular junction-dependent signal amplification mediated by single ribonucleotide repair: detection of DNA methyltransferase (MTase) involves two sequential reaction steps. First, dilute all oligonucleotides with 1× Tris-EDTA buffer (10 mmol / L Tris, 1 mmol / L ethylenediaminetetraacetic acid (EDTA), pH 8.0) Acid sequence to prepare stock solutions. Then, the hairpin substrate and ligation probe 2 (LP2) were dissolved in buffer (1.5 mmol per liter of magnesium chloride, 10 mmol per liter of tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl), pH 8.0) to 10 micromolar per liter and incubated at 95°C for 5 minutes, then slowly cooled to room temperature to form a hairpin structure. Subsequently, 1 µl of hairpin substrate (10 µmol / L) was added to 20 µl containing Dam MTase, 160 µmol / L of SAM, 6 units of Dpn I, 2 µl of 10×Dam MTase Reaction buffer and 2 μl of 10×CutSmart buffer were incubated at 37°C for 2 hours, then inactivated at 80°C for 20 minutes. Then 5 mic...

Embodiment 2

[0088] (1) Experimental verification of the principle:

[0089] In order to verify whether Dam MTase can cleave hairpin substrates in the presence of Dpn I, we used non-denaturing polyacrylamide gel electrophoresis and used SYBR Gold as a fluorescent indicator to analyze the reaction products ( figure 2 A). When both Dam MTase and Dpn I do not exist ( figure 2 A, lane 1), only Dam MTase ( figure 2 A, lane 2) and only Dpn I ( figure 2 When A lane 3) was present, only one 56-nt band was observed, which was the same size (56 nt) as the synthetic hairpin substrate, indicating that no methylation and cleavage reactions occurred. But when in the presence of Dam MTase and Dpn I, a new 24-nt band was observed ( figure 2 A, lane 4), indicating that Dam MTase can trigger the methylation of the hairpin substrate, and then the methylated hairpin substrate can be cleaved by Dpn I at the methylated adenine site.

[0090] In order to verify the cyclic junction-dependent SDA reactio...

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Abstract

The invention discloses a method for detecting the activity of DNA methyltransferase based on single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification. The method comprises the following steps: (1) adding a Dam MTase acted hairpin substrate to a resection reaction buffer solution, and carrying out a cleavage reaction to produce a 24-nt product; and (2) adding the cleavage product to an amplification reaction buffer solution, carrying out a cyclic linkage-dependent strand displacement amplification reaction to cyclically generate an enhanced fluorescent signal, anddetecting the intensity of the fluorescent signal to determine the activity of the Dam MTase. The method can effectively inhibit nonspecific amplification independent of a target and a template / primer, so the background signal is greatly reduced, and the index amplification of the signal is finally achieved. The method has a high sensitivity and a high specificity, and the lower detection limit can reach 4.8 * 10<-6> U / mL.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a method for detecting DNA methyltransferase activity based on single ribonucleotide repair-mediated connection-dependent cycle signal amplification. Background technique [0002] Genomic DNA methylation often occurs at the carbon 5 / nitrogen 4 position of cytosine (C) and the nitrogen 6 position of adenine (A), which is the most important epigenetic modification in genomic DNA. Plays a crucial role in regulating gene transcription, chromatin structure, embryonic development and cellular aging. DNA methyltransferase (MTase) is responsible for the methylation modification of genomic DNA, which can catalyze the transfer of methyl groups on adenosyl-L-methionine (SAM) to adenine / cytosine residues in specific genomic DNA sequences , to maintain a stable methylation pattern in the cell. Abnormal DNA methyltransferase (MTase) activity can destroy the normal DNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/48C12Q1/44
CPCC12Q1/44C12Q1/48C12Q1/6844G01N2333/91011G01N2333/922C12Q2531/119C12Q2525/301C12Q2521/501C12Q2521/327
Inventor 张春阳王黎娟韩笑
Owner SHANDONG NORMAL UNIV
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