Comparative Analysis Of Real-time Quantitative PCR-Sanger Sequencing Method And Taqman Probe Method For Detection Of K-RAS/B-RAF Mutation In Colorectal Carcinomas

Objective To analyze comparatively the application value of two different methods fordetecting the K-ras/B-raf mutations; To detect the correlations of K-ras/B-raf mutations to theclinical pathological characteristics in colorectal carcinomas (CRC) patients; To investigatemutations of the K-ras/B-raf and its relationship to risk assessment.Method Genomic DNA was extracted from formalin fixed paraffin embedded (FFPE)samples of344CRC from the tumor cells by the micro-dissection. Real-time quantitativePCR-Sanger sequencing method and Taqman probe method were performed to detect theK-ras/B-raf mutations. To analyze the correlations to the frequency and types of K-ras/B-rafmutations, and clinical pathological characteristics and survival time.Results K-ras mutations were detected in39.8%(137/344) and38.7%(133/344) of344CRCsby using Real-time quantitative PCR-Sanger sequencing method and Taqman probe methodrespectively. B-raf mutation was detected in4.7%(16/344) and4.1%(14/344) respectively. Therewas no significant correlation between these two methods for the positive rate of K-ras/B-rafmutation. The frequency of the K-ras mutation in female was higher than that in male(P＜0.05).The frequency of the B-raf mutation in colon was higher than that in rectum. The frequency ofthe B-raf mutation in Ⅲ-Ⅳ stages was higher than that in Ⅰ-Ⅱ stages. The frequency of theB-raf mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma andnon special type adenocarcinoma was the lowest. The frequency of the B-raf mutation in Ⅲgrade was higher than that in Ⅱ grade(P＜0.05). The overall concordance for the two methodsof K-ras/B-raf mutation detection was98.8%(kappa=0.976). There were statistics significancebetween B-raf and K-ras mutation for the survival time of CRC(P=0.039). There were statisticssignificance between B-raf mutation type and B-raf/K-ras wild type(P=0.058).Conclusion (1) In our study, comparing with the Real-time quantitative PCR-Sangersequencing method, Taqman probe method is better for those including handling time, efficiency,repeatability, cost and equipment;(2) In our study, the frequency of the K-ras mutation iscorrelated with gender. The B-raf mutation was associated with primary tumor site, TNM stage,histological types and histological grades;(3) In our study, B-raf gene mutation was anindependent prognostic mark for the CRC.