Method of detecting UDG activity by enzyme-mediated two-step serial signal amplification based on excision repair

Abstract

The invention discloses a method of detecting the UDG activity by enzyme-mediated two-step serial signal amplification based on excision repair. The method comprises the following steps: (1) adding a UDG active substrate in an excision reaction buffer solution to carry out excision repair reaction, excising a uracil base of the UDG active substrate, and leaving an abasic site; and (2) adding an excision repair reaction product in an amplification reaction buffer solution to carry out enzyme-assisted two-step serial signal amplification reaction, namely inducing strand displacement reaction and index amplification reaction, generating strengthened fluorescence signals in a circulation manner, and determining the UDG activity through weakness of the fluorescence signals. According to the method provided by the invention, ultrahigh sensitive detection to the activity of uracil DNA glycosylase (UDG) is realized by utilizing high amplification efficiency of constant-temperature index amplification reaction and specific circulation digestion of ribonuclease H.

Description

Technical field [0001] The present invention relates to the technical field of bioanalysis, in particular to a method for detecting UDG activity based on excision repair and enzyme-mediated two-step series signal amplification. Background technique [0002] The genome is composed of specifically paired A:T / G:C bases, and its accuracy and stability are of great significance to the maintenance of life. But in real life, the structure of genomic DNA bases often suffers from external factors (such as ultraviolet rays, ionizing radiation, toxic chemicals) and endogenous active factors (such as superoxide anions, hydroxyl radicals, hydrogen peroxide, etc.) The damage caused by DNA single-strand and double-strand breaks, mismatches, base deletions, etc., destroys the integrity of the genome, causes genome instability, induces cancer, and affects the survival of species. In order to reduce the damage of genomic DNA in the body, a variety of damage repair mechanisms have evolved in organ...

AI Technical Summary

Problems solved by technology

Among them, the colorimetric analysis method uses hydrogen peroxide to oxidize 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS 2- ) produces colored ABTS - , to visualize the activity of uracil-DNA glycosylase (UDG), but its detection sensitivity cannot be compared with electrochemical and fluorescent methods

The electrochemical analysis method improves the detection sensitivity to a certain extent, but it needs to immobilize the capture probe on the solidified carrier and prepare the graphene electrode in advance, which is time-consuming and laborious.

The fluorescence method is simple in design and easy to operate, but it cannot avoid the disadvantages of low detection sensitivity and unstable signal

In summary, although these methods for detecting uracil DNA glycosylase (UDG) activity are effective, they still have shortcomings such as low sensitivity, long loss time, and cumbersome operations.

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