Electronic Microarray Technique for Detection of Nine Base Substitutions Including Single-Nucleotide Polymorphisms in the Human OGG1 Gene

摘要

Single-nucleotide polymorphisms (SNPs) are potentially important biomarkers for molecular diagnostics. Several SNPs and somatic mutations have been characterized in a human 8-hydroxyguanine glycosylase gene, OGG1 , which codes for a DNA repair enzyme responsible for excising 8-hydroxyguanine from damaged DNA (1)(2). The distribution of genotypes for a SNP, S326C, in the OGG1 gene has been implicated in the susceptibility to various cancers, including lung, esophageal, and prostate cancer (2)(3)(4). A functional consequence of this SNP is a difference in the activity to suppress mutagenesis induced by 8-hydroxyguanine. Another SNP, R46Q, causes alternative splicing as well as a difference in the mutation suppressive activity. In addition, somatic mutations in the OGG1 gene have been detected in a subset of lung, gastric, and kidney cancers, suggesting that aberrations of OGG1 contribute to human carcinogenesis by enhancing mutagenesis induced by 8-hydroxyguanine (2)(3)(4)(5).The objective of this study was to develop a novel technique that can provide multiplex analysis of base substitutions including SNPs in the OGG1 gene by use of electronic microarray technology. Here we report the development of an electronic microarray technique for analysis of all nine base substitutions in 1-kb amplicons. The technique was successfully applied to the analysis of the four SNPs and five mutations in the OGG1 gene in four cancer cell lines with a double-blind experimental design, and the results were in excellent agreement with the previous identification of SNPs in the cell lines by DNA sequencing and single-strand conformation polymorphism gel electrophoresis. For quality control, we used site-directed mutagenesis and megaprimer PCR techniques (6)(7) to develop a 1-kb DNA consisting of all base substitutions in the OGG1 gene. The present technique has the unique capability of simultaneous detection of multiple base substitutions …