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Application of DNA three-way regulation activation based hybridization chain reaction to high sensitivity detection of DNA methyltransferase

A hybrid chain reaction and methylation technology, applied in the field of enzyme activity detection, can solve problems such as lack of recognition mechanism, and achieve good specificity.

Active Publication Date: 2015-11-25
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the substrate for DNA modifying enzymes is a DNA sequence or a specific base site. The biggest obstacle to using DNA modifying enzymes to construct DNA nanodevices is the lack of an effective recognition mechanism. How to convert the action of such enzymes into DNA Three-way joint activation process is a difficult problem faced by those skilled in the art

Method used

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  • Application of DNA three-way regulation activation based hybridization chain reaction to high sensitivity detection of DNA methyltransferase
  • Application of DNA three-way regulation activation based hybridization chain reaction to high sensitivity detection of DNA methyltransferase
  • Application of DNA three-way regulation activation based hybridization chain reaction to high sensitivity detection of DNA methyltransferase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment one, experimental method

[0038] The role of M.SssI and its recognition sequence

[0039] Take different concentrations of M.SssI, 160μMSAM and methyltransferase recognition probe and mix with NEBuffer2 (1×), react for 2 hours, add methylation-sensitive restriction endonuclease HpaII and mix with Cutsmart (1×) buffer , Reaction 2h. For HpaII inactivation, the inactivation condition is to keep at 80° C. for 30 minutes, and lower the temperature slowly.

[0040] Performing a hybridization chain reaction

[0041] In the above system, add 1×TNaK buffer solution, H1, H2 and ultrapure water, vortex and mix well and react for 2 hours. Maintain the complete methyltransferase recognition sequence and mix with H1 and H2 for hybridization chain reaction.

[0042] Dye addition and fluorescence measurement

[0043] After the above hybridization chain reaction is completed, add NMM, KCl and ultrapure water to the reaction system to increase its volume to 50 μL. Afte...

Embodiment 2

[0047] Example 2. The hybridization chain reaction based on DNA three-way joint activation is used for the highly sensitive detection of DNA methyltransferase

[0048] The Principle of Hybridization Chain Reaction Based on Activation of DNA Three-way Knot for Detection of M.SssI Activity

[0049] In order to achieve the generality of this method, we designed a novel DNA three-way junction activation method based on the protection mechanism of restriction endonucleases for the determination of CpG methyltransferase activity. Specific principles such as figure 1 Shown: The recognition probe (RMP) of M.SssI is formed by direct annealing of a DNA strand carrying a Toehold part and a DNA strand carrying a strand migration part. In RMP, the Toehold part and the strand migration part are close to each other, so they can act as the priming strand to trigger DNA assembly. In addition, its stem contains recognition sequences and sites for restriction endonucleases HpaII and M.SssI. W...

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Abstract

The present invention relates to application of DNA three-way regulation activation based hybridization chain reaction to high sensitivity detection of DNA methyltransferase. The present invention uses DNA methyltransferase M.SssI as a model to develop an enzyme protection cleavage recognition mechanism, which converts the effect of enzyme on a substrate into a trigger process of a hybridization chain reaction, thereby causing a DNA self-assembly process and achieving construction of a universal DNA nanodevice. The device can be applied to high sensitivity detection of DNA modification enzyme (especially DNA methyltransferase) and inhibitor screening, and has potential application value.

Description

technical field [0001] The invention relates to the field of enzyme activity detection, in particular to the highly sensitive detection of DNA methyltransferases by hybridization chain reaction activated by DNA three-way joints, in particular to the detection of DNA methyltransferase M.SssI. Background technique [0002] In the past decade, DNA has become an interesting assembly element for the controllable and programmatic design and assembly of DNA nanomaterials due to its strict base-pairing principle. Through rational sequence design and DNA self-assembly process, a series of dynamic DNA nanodevices have been successfully assembled, such as nanomachines, logic gates, catalytic amplifiers, etc., most of them are based on Toehold-mediated strand displacement reactions. DNA nanodevices are usually initiated by pH adjustment, nucleic acid hybridization, and the reaction of metal ions with bases. However, the general design only converts one target into signal output, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/44
Inventor 姜玮王磊朱静
Owner SHANDONG UNIV
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