Method for detecting human 8-hydroxyl guanine DNA glycosylase activity through circular signal amplification based on autocatalytic replication mediation

Abstract

The invention discloses a method for detecting human 8-hydroxyl guanine DNA glycosylase activity through circular signal amplification based on autocatalytic replication mediation. A biological sensorcomprises a hairpin substrate, MB1, MB2, Fok I and APE1. A damaged guanine and palindromic sequence is designed in a sense strand or an antisense strand of the hairpin substrate, a damaged guanine islocated between the palindromic sequence and a ring, and a cytosine matched with the damaged guanine is designed at the other strand; MB1 is of a stem ring structure with the protruding 5' end, the palindromic sequence is designed at the ring of MB1, a sequence complementary for the ring of the hairpin substrate is designed on the 5'-tail end DNA sequence protruding from MB1, and fluorescent molecules and quenching molecules are modified on the MB1 and the MB2. The method can achieve easy, convenient, high-sensitive and high-specific detection of hOGG1.

Description

technical field [0001] The invention relates to biological analysis technology, in particular to a method for detecting human 8-hydroxyguanine DNA glycosylase activity based on self-catalyzed replication-mediated cycle signal amplification. Background technique [0002] The statements herein merely provide background information related to the present invention and may not necessarily constitute prior art. [0003] Genomic DNA is composed of Watson-Crick paired heterocyclic bases (ie, A and T and G and C), which is the main carrier of genetic information and is responsible for translating the genetic code into multifunctional proteins. The maintenance of genomic DNA accuracy and integrity is a fundamental prerequisite for the survival of all living organisms. However, in real life, genomic DNA is often damaged by various exogenous and endogenous factors, resulting in the existence of at least 10 4 Various damages (such as base oxidation, DNA alkylation, DNA deamination, DN...

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Problems solved by technology

However, the above methods involve expensive fluorescent nanomaterials (QDs), sophisticated instruments (i.e., total internal reflection fluorescence microscopy (TIRFM)), and complex operations (i.e., TIRF-based single-molecule imaging), which greatly limits their wide application.

In addition, exonuclease III-induced recycling signal amplification and lambda exonuclease-assisted recycling signal amplification techniques were used to measure the activity of hOGG1 and uracil-DNA glycosylase (UDG) fluorescently, although detection Sensitivity is improved, but they all suffer from high background signal caused by nonspecific cleavage by exonucleases

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