8-oxoguanine DNA glycosylase determination method based on background signal inhibition probe, kit and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an 8-oxoguanine DNA glycosylase determination method based on a background signal inhibition probe, a kit and an application thereof. According to the present invention, the selectivity of Lambda exonuclease on the substrate structure and the recognition cutting effect of 8-oxo guanine DNA glycosylase on 8-oxo guanine are utilized, and the inhibition effect of the background signal inhibition probe on the lambda exo side activity is combined so as to construct the new 8-oxo guanine DNA glycosylase fluorescence determination method. The method specifically comprises the following steps: mixing a lambda exo buffer solution, a reporter probe sequence containing a fluorophore and a quenching group, and a background signal inhibition probe sequence containing 8-oxo guanine; heating the mixed solution to 85 DEG C, then gradually cooling to 37 DEG C, and adding a sample to be detected; incubating at 37 DEG C; and after incubation is finished, adding lambda exo, carrying out enzyme digestion reaction, and then carrying out fluorescence detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for measuring 8-oxoguanine DNA glycosylase based on a background signal suppression probe, a kit and an application thereof. Background technique [0002] 8-Oxoguanine is a common DNA oxidative damage caused by reactive oxygen species, which can easily cause gene mutation and seriously affect the health of living organisms. 8-oxoguanine DNA glycosylase (8-oxoguanine DNAglycosylase, OGG) is a key enzyme in the process of DNA repair. 8-oxoguanine DNA glycosylase has both N-terminal glycosylase activity and AP (apurinic / apyrimidinic)-lyase activity. N-terminal glycosylase activity can cleave damaged purine bases on double-stranded DNA, creating an AP site. AP-lyase activity cleaves either the 3' or 5' end of the AP site, creating a base gap with 3' and 5' phosphates. 8-oxoguanine DNA glycosylase can recognize and excise 8-oxoguanine, which is of great significanc...

AI Technical Summary

Problems solved by technology

However, the existing fluorescence methods have a problem that the sensitivity of the method is always directly proportional to the complexity of the detection system

Fluorescence detection methods with high sensitivity often need to use a variety of expensive materials, resulting in high detection costs and complicated operation procedures

For example, applications using quantum dots can achieve a limit of detection (LOD) as low as 1.8×10 -3 U / mL, but the preparation process of quantum dots is complicated and difficult for practical application; while the sensitivity of simple detection systems is often difficult to meet the detection requirements

Existing fluorescent detection methods for 8-oxoguanine DNA glycosylase cannot balance low cost and high sensitivity

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