High-sensitivity uracil DNA glycosylase (UDG) detection using DNA three-direction section activated hybridization chain reaction

A technology of hybrid chain reaction and glycosylase, which is applied in the field of enzyme activity detection, can solve the problems of lack of recognition mechanism and achieve good specificity and low detection limit

Active Publication Date: 2015-12-09
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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  • High-sensitivity uracil DNA glycosylase (UDG) detection using DNA three-direction section activated hybridization chain reaction
  • High-sensitivity uracil DNA glycosylase (UDG) detection using DNA three-direction section activated hybridization chain reaction
  • High-sensitivity uracil DNA glycosylase (UDG) detection using DNA three-direction section activated hybridization chain reaction

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment one, experimental method

[0041] UDG and its recognition sequence interaction

[0042] Add the annealed UDG recognition sequence, different concentrations of UDG enzyme, UDG action buffer and ultrapure water into 10 μL reaction system, and react for 45 minutes after vortex mixing.

[0043] Performing a hybridization chain reaction

[0044] After UDG reacts with its recognition sequence, the conformational transition of the hairpin occurs, and the toehold and the chain migration part originally enclosed in the rigid structure of the hairpin get close to form a complete hybridization chain reaction initiation chain. In the above system, add 1×TNaK buffer solution, H1, H2 and ultrapure water, vortex and mix well and react for 2 hours.

[0045] Dye addition and fluorescence measurement

[0046] 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. After vortex mixin...

Embodiment 2

[0050] Example 2: Highly sensitive detection of uracil DNA glycosylase by hybridization chain reaction based on DNA three-way joint activation

[0051] The principle of detection of uracil DNA glycosylase activity by hybridization chain reaction based on DNA three-way joint activation

[0052] The invention designs the hairpin remodeling strategy induced by UDG enzyme, and combines the isothermal amplification function of the hybridization chain reaction to carry out specific recognition and high-sensitivity detection. The specific principle is as follows: UDG recognition probe (RUP) is designed, which includes UDG recognition sequence, toehold, strand migration part and two complementary auxiliary sequences. When UDG enzyme is added to the system, the U on the recognition probe is removed to generate an abasic site (APsite), which makes the hybridization of the chain migration part and the UDG recognition sequence extremely unstable. At this time, the complementary auxiliary...

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Abstract

The invention relates to high-sensitivity uracil DNA glycosylase (UDG) detection using DNA three-direction section activated hybridization chain reaction. UDG is used as a model to develop an identification mechanism of hairpin reconstruction, and action of DNA modification enzyme on substrate is converted into the triggering process of the hybridization chain reaction so as to cause the self-assembling process of DNA and build a universal DNA nano device. The device can be used for the high-sensitivity detection and inhibitor screening of the DNA modification enzyme (especially the UDG), 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 uracil DNA glycosylase by hybridization chain reaction activated by DNA three-way joint. 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 greatly limits the diversified design of target-responsive DNA na...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/34
Inventor 姜玮王磊朱静
Owner SHANDONG UNIV
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