A method for measuring 8-oxoguanine DNA glycosylase based on background signal suppression probe, kit and application thereof

Abstract

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. The present invention utilizes the selectivity of Lambda exonuclease to the substrate structure and the recognition and cleavage of 8-oxoguanine by 8-oxoguanine DNA glycosylase, combined with the background signal to suppress the effect of the probe on the side activity of λexo Inhibition, a new 8‑oxoguanine DNA glycosylase fluorescence assay method was constructed. Specifically include: mixing λexo buffer, reporter probe sequences containing fluorescent groups and quenching groups, and background signal suppression probe sequences containing 8-oxoguanine; heating the mixture to 85°C, and then cooling down gradually Add the sample to be tested at 37°C; incubate at 37°C; after the incubation, add λexo to carry out enzyme digestion reaction, and then perform 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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