A method for detecting DNA glycosylase activity based on single quantum dot level

Abstract

The invention discloses a method for detecting DNA (deoxyribonucleic acid) glycosylase activity on basis of single quantum dot level.In detection, DNA glycosylase hOGG1 specifically recognizes and excises damaged guanine to leave an abasic site, apurinic endonuclease-1 further excises the abasic site to leave a nucleotide gap, and DNA polymerase beta polymerizes Cy5-dGTP at the gap to generate a double-tagging double-strand nucleotide substrate; by specific reaction between biotin and streptavidin, the DNA substrate is combined to the surface, covered with the streptavidin, of quantum dots to form a QD-DNA-Cy5 compound; due to reduction of spatial distance, fluorescence resonance energy transfer occurs between the quantum dots and Cy5, and Cy5 fluorescence signals can be observed in the unimolecular level.The method has the advantages of simplicity, quickness and sensitivity, and the lower limit of detection can reach 1.8*10<-6>U / microliter.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a method for detecting DNA glycosylase activity based on a single quantum dot level. Background technique [0002] The maintenance of genomic DNA integrity is of great significance to the stability of species, but in real life, genomic DNA is inevitably affected by various factors from inside and outside the body, exogenous sources such as radiation (UV), chemical mutagens etc., endogenous ones such as reactive oxidizing substances (ROS), etc. These factors can lead to DNA single- and double-strand breaks, mismatches, and base deletions, thereby destroying the integrity of the genome and affecting the survival of species. In order to reduce DNA damage, organisms have formed a variety of damage repair mechanisms during evolution, including mismatch repair (MMR), base excision repair (BER), and nucleotide excision repair (NER) for single-base mutations. Amo...

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Problems solved by technology

These detection methods are very effective, but very time-consuming, cumbersome to operate, and have potential safety hazards

[0004] In order to overcome the above shortcomings, in recent years, colorimetric detection methods based on nanotechnology and fluorescent probe detection methods based on various fluorescent dyes have also been developed. Highly sensitive detection of uracil DNA glycosylase, using DNA glycosylase UDG as a model, developed the recognition mechanism of hairpin remodeling, and transformed the action of DNA modifying enzymes on substrates into the triggering process of hybridization chain reaction , thereby causing the self-assembly process of DNA, and realizing the construction of a universal DNA nano-device, which can be used for highly sensitive detection of DNA modifying enzymes (especially UDG), wherein the detection limit of UDG reaches 0.000043U / mL; CN104630363A A method for detecting uracil-DNA glycosylase activity based on a label-free, enzyme-free DNA machine fluorescence amplification strategy is disclosed. A double-stranded DNA (dsDNA) probe containing uracil bases and a priming sequence is used to identify UDG targets and With the release of the priming strand, the priming strand can activate the label-free enzyme-free DNA machine to generate an amplified fluorescent signal. Due to the design of the dsDNA probe and the specific DNA machine, the detection method successfully achieves background reduction and signal amplification, resulting in low detection limit (0.00044U / mL); however, nanoparticle processing is cumbersome, time-consuming, complex operation and low detection sensitivity, while fluorescence detection methods often have the disadvantages of difficult design of fluorescent probes and high cost, and limited improvement in detection sensitivity

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