The Experimental Study On Detection Of K-ras Mutation Through PNA-clamp Mediated Real-time PCR In Colorectal Cancer

Objectives:This study was designed to evaluate the effect of direct sequencing(Sanger method) and PNA-clamp mediated real-time PCR for detecting the K-ras gene mutations in colorectal cancer and adjacent tissues, investigating the clinical value of K-ras gene mutations, and exploring the relationship between K-ras gene mutation and the clinicopathological factors of colorectal cancer.Methods:This study collected fresh tumor tissues, adjacent tissues from35colorectal cancer patients while accepting operation in Xiangya Hospital of Central South University between Jan,2012and Dec,2012.Each patient’s serum sample was collected before the operation. The DNA was extracted by the kit method, then the most7common mutation types of K-ras gene in codon12and codon13of exon2was assayed by Sanger sequencing and peptide nucleic acid-clamp mediated real-time PCR methods. At last, the results combined with the clinical data were analyzed.Results:(1)9K-ras gene mutations were detected by using Sanger method among the35colorectal cancer tissues. The positive rate was25.7%,including7mutations in codon12:35G>A in3patients,35G>T in3patients and34G>T in1patients, and2mutations in codon2. None of K-ras gene mutation was detected in the adjacent tissues of colorectal cancer patients.14K-ras gene mutations were detected by using PNA-clamp mediated real-time PCR from the35colorectal cancer tissues. The positive rate was40.0%. The difference between anger method and PNA-clamp mediated real-time PCR for detecting K-ras gene mutation in colorectal cancer reached statistical significance.2K-ras gene mutations were also detected in adjacent tissues of colorectal cancer by PNA-clamp mediated real-time PCR, but the mutation ratio was significantly lower than the colorectal cancer.(2)4K-ras gene mutations were detected in the serum of35colorectal cancer patients by PNA-clamp mediated real-time PCR. The positive rate was11.4%. Kappa consistency check method was used to analyze the tumor tissues gene mutation results data and serum gene mutation results data detected by PNA-clamp mediated real-time PCR. The Kappa value is0.189.(3)There was no significant correlation between K-ras gene mutation rate and age, gender, histological type, tumor location, TNM staging of colorectal cancer.Conclusions:(1) Comparing with the K-ras gene mutation result detected by Sanger method in colorectal cancer patients, PNA-clamp mediated real-time PCR has a higher sensitivity and higher detection rate,which is more suitable for clinical application.(2) Small consistency was found after the data analysis of gene mutation results detected by serum and gene mutation results detected by tissue of colorectal cancer patients by PNA-clamp mediated real-time(3) No significant correlation war found between the common clinical pathological factors and K-ras gene mutation rate in our study.