Single nucleotide polymorphism detection in the hMSH2 gene using conformation-sensitive CE.
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 degrees 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.
Chen
Yen-Ling
YL
Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.
Jong
Yuh-Jyh
YJ
Ferrance
Jerome
J
Hsien
Jan-Sing
JS
Shih
Chi-Jen
CJ
Feng
Chia-Hsien
CH
Wu
Ming-Tsang
MT
Wu
Shou-Mei
SM
D001483:Base Sequence; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011108:Polymers; D020641:Polymorphism, Single Nucleotide; D013696:Temperature; D014508:Urea
D017931:DNA Primers; D004273:DNA, Neoplasm; D011108:Polymers; D014508:Urea; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein