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Establishment of pyrosequencing method for detection of k-ras mutation

AIM:To establish a pyrosequencing method for detecting k-ras mutation and to investigate the frequency and characteristic of k-ras of patients with colorectal cancer.METHODS:A total of 10 DNA specimens from the ministry of health clinical test center of China for external quality assessment were prepared for k-ras mutation detecting method verification.A total of 230 DNA specimens from patients with colorectal cancer were used for kras mutation detection.RESULTS:The overall positive rate of k-ras gene mutations obtained by pyrosequencing was 14.34%(33 /230),and the positive rate of mutations was 10%(23 /230) at codon 12[5.22%(12 /230) for 35 G A,3.04%(7 /230)for 35 G T,1.74%(4 /230) for 34 G T] and4.34%(10 /230) at codon 13[0.43%(1 /230)for 37 G A,3.91%(9 /230) for 38 G A].CONCLUSION:This pyrosequencing method to detect k-ras mutation is proved to be a rapid,accurate and high throughput method alternative to conventional method,and it can be a preferred option in research and clinical application.