环境化学污染物暴露相关基因甲基化焦磷酸测序方法的构建和应用

doi:10.3969/j.issn.1004-616x.2017.02.001

Abstract

Abstract:

OBJECTIVE: To establish a pollutant-responsive gene-specific pyrosequencing assay for quantitative measurement of DNA methylation in peripheral blood lymphocytes of coke-oven workers. METHODS: We firstly analyzed DNA methylation levels of RAS-associated domain family 1 (RASSF1A) in 69 coke-oven workers and 46 controls using bisulfate sequencing method. The hyper-methylated hotspots in the exposure group were recognized as the target region for design of specific primers of pyrosequencing. As a pilot study,we examined the methylation status of RASSF1A in 11 paired coke-oven workers and controls. RESULTS: Pyrosequencing was the optimized assay for DNA methylation detection. Compared to BSP assay,pyrosequencing exhibited advantages in efficiency,simplicity,accuracy and economical performances. We were able to distinguish occupational exposed workers from controls effectively using this method. The methylation level of RASSF1A in coke-oven workers was increased by 87.15% compared to that in the controls (9.32%±3.82% and 4.98%±2.28%,respectively,P < 0.01). CONCLUSION: Compared to the BSP assay, gene-specific pyrosequencing method exhibited strength in specificity and accuracy of methylation detection of environmental pollutant-responsive gene in PAHs-exposed workers.

Key words: pyrosequencing, PAHs, occupational exposure, DNA methylation, biomarkers

CLC Number:

R114

ZHANG Haiyan, LI Qingye, CHEN Shen, CHEN Wen, HE Zhini. Construction of an environmental pollution-responsive gene-specific pyrosequencing assay[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2017, 29(2): 81-86.

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Construction of an environmental pollution-responsive gene-specific pyrosequencing assay

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