Method for jointly detecting whole genome DNA adduct based on dot blot hybridization and chromatin co-immunoprecipitation sequencing

Abstract

The invention discloses a method for jointly detecting a whole genome DNA adduct based on dot blot hybridization and chromatin co-immunoprecipitation sequencing. The method adopts the dot blot hybridization technology to detect the total amount of the DNA adduct and optimize each test condition so as to obtain the DNA adduct state of the whole genome in the maximum range, then the chromatin co-immunoprecipitation sequencing technology principle is introduced, a DNA fragment library of a BPDE-DNA adduct is generated in an established contaminated cell by using a specific recognition reaction of an antigen and an antibody, an injury map is obtained by a sequencing technology, the site of the DNA adduct in a whole genome is defined so as to screen a gene related to lung cancer caused by BPDE, and the gene related to lung cancer caused by BPDE is screened by combining a BPDE-DNA adduct antibody and specifically enriching a DNA fragment acted by BPDE for sequencing, so that more comprehensive and exact information is provided for further screening key pathways and targets of B[a]P carcinogenesis.

Description

technical field [0001] The invention relates to the technical field of detection and identification of BPDE adducted genes, and more specifically, relates to a method for joint detection of whole-genome DNA adducts based on dot hybridization and chromatin immunoprecipitation sequencing. Background technique [0002] Benzo[a]pyrene (B[a]P), as one of the common polycyclic aromatic hydrocarbons in tobacco, smoke and the environment, is a typical DNA damage carcinogen. Due to its strong mutagenic and carcinogenic effects, It is listed as one of the monitoring items of environmental pollution at home and abroad. Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) is B[a] The final metabolite of P, which can covalently bind to DNA, form apurinic adducts with guanine, and phosphotriester adducts with the DNA phosphate backbone, thereby destroying the structure and function of biological macromolecules such as DNA and proteins . The active metabolite is covalently combined with D...

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Problems solved by technology

Only Chinese patent CN 110066863 A discloses a method based on the principle of chromatin immunoprecipitation technology, which uses Anti-BPDE antibody to perform high-throughput sequencing of enriched BPDE-DNA adducts to construct B[a]P adducts The precise positioning technology of the target gene locus is to infect the cells by selecting some fragments of the lysed cells after the cells are lysed, which cannot reflect the real BPDE-DNA adduct formation, and because various treatments in the process It will affect the total amount of BPDE-DNA adducts in the cells. In the absence of detection and evaluation methods for the total amount of DNA adducts, it is easy to cause the loss of DNA adducts, resulting in information loss, and cannot be more comprehensive Revealing B[a]P damage to genome-wide DNA sequences

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