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Single nucleotide polymorphism detection in the hMSH2 gene using conformation-sensitive CE

Author:
Chen, Yen-Ling, Jong, Yuh-Jyh, Ferrance, Jerome, Hsien, Jan-Sing, Shih, Chi-Jen, Feng, Chia-Hsien, Wu, Ming-Tsang, Wu, Shou-Mei
Source:
Electrophoresis 2008 v.29 no.3 pp. 634-640
ISSN:
0173-0835
Subject:
DNA, DNA repair, cellulose, colorectal neoplasms, electrophoresis, genes, genotype, humans, nucleotides, patients, polymerase chain reaction, promoter regions, screening, sequence analysis, single nucleotide polymorphism, solvents, temperature, urea, volunteers
Abstract:
CE allows for highly reproducible analysis of DNA fragments which can be used to detect DNA mutations including SNPs. We have utilized a simple and direct CE analysis method for SNP analysis called conformation-sensitive CE (CSCE), based on the principle of single nucleotide different to produce conformational changes in the mildly denaturing solvent system. This method was applied to analysis of a mutation in the promoter region of the hMSH2 gene. This gene belongs to the human DNA mismatch repair system, which is responsible for recognizing and repairing mispaired nucleotides, and mutations in the hMSH2 gene are known to cause hereditary nonpolyposis colorectal cancer (HNPCC). PCR fragments generated from the promoter region of the hMSH2 gene, displaying either a C/C homozygote, C/T heterozygote, or T/T homozygote genotype, did not require further pretreatment before electrokinetic injection. The CE separation, using a 1xTris-borate-EDTA (TBE) buffer containing 3% w/v hydroxylethyl cellulose (HEC) and 6 M urea, was performed under reverse polarity with a separation temperature of 15°C. The genotypes of 204 healthy volunteers and 13 colorectal cancer patients were determined using CSCE, and the results confirmed by DNA sequencing. While the CSCE separations were shown to be highly reproducible and sensitive for screening large populations, no correlation was observed between cancer patients and this hMSH2 gene polymorphism.
Agid:
2068541