Peptide nucleic acid-mediated one-step PCR assay in detection of K-ras mutation

Abstract

Objective:To establish a peptide nucleic acid-mediated one-step PCR assay for detecting K-ras mutation,and to evaluate its diagnostic value.Methods: We developed a one-step polymerase chain reaction (PCR) approach with melting curve analysis using wild-type specific peptide nucleic acid (PNA) and fluorescent dye SYBR Green Ⅰ to determine the genotypes in codon 12 and 13 of K-ras oncogene. The result of our method was compared with that of restriction fragment length polymorphism (RFLP) analysis.Results: Our method simultaneously examined condon 12 and 13 of K-ras oncogene,and was easy to perform.The sensitivity of our method was 0.001% when in a 105-fold excess of wild-type K-ras DNA.The detection limit was 102 copies.Our method could be used for large sample test,with the time of a single test being 1 hour.The limit of traditional method was 103copies and the sensitivity was 0.01%.The testing time was about 2 days for samples which needed only 1 hour using our method.Conclusion: Our method has obvious advantage over RFLP analysis; it is suitable for detecting K-ras mutation in large samples and can be used as an effective method for early diagnosis of pancreatic cancer.