Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis.
Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1x Tris-borate-EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250,000) under reverse polarity with 15 degrees C and 30 degrees C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.
Chen
Yen-Ling
YL
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Chang
Ya-Sian
YS
Chang
Jan-Gowth
JG
Wu
Shou-Mei
SM
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D003062:Codon; D015179:Colorectal Neoplasms / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D017354:Point Mutation; D018631:ras Proteins / Q000235:genetics*
D003062:Codon; D018631:ras Proteins