Peptide Nucleic Acid-mediated PCR Clamping: A Novel K-ras Mutation Detection Assay For Plasma Samples Of Patients With Pancreatic Cancer

Human pancreatic ductal adenocarcinoma is a devastating cancer that results in approximately the same number of deaths on an annual basis. As such, it carries with it the worst prognosis of any gastrointestinal cancer with median survival of 6 months. Five-year survival for all stages of pancreatic cancer is less than 5%, although this is on the order of 20% for early stage pancreatic cancers subjected to surgery with intent to cure. For early diagnosis,effective new markers of pancreatic carcinoma are urgently needed. Gene alterations of the ras type can be used as molecular markers to screen for the presence of neoplastic cells in different clinical specimens. Many conventional methods are able to discriminate mutant from wild-type alleles and also provide a high degree of sensitivity to detect mutant alleles in a large excess of normal DNA. Two PCR-based techniques have been most commonly used: ASA-PCR (allele-specific amplification), which is restricted to the analysis of individual codon-specific mutations and requires several allele-specific oligonucleotides, and the time-consuming RFLP-PCR (restriction fragment length polymorphism), which bears an increasing risk of Taq polymerase-born infidelity. More recently, the mutation-sensitive hybridisation profile of peptide nucleic acids (PNAs) has been exploited to design novel protocols called PCR-clamping. In this study, we combined the PCR-clamping approach with melting curve analysis using wild-type specific PNAs to determine the genotypes of the most frequent point mutation in codon 12 and 13 of K-ras in blood samples of patients. The sensitivity of our assay, includes 67 patients of pancreatic cancer(PC) and 22 patients of chronic pancreatitis(CP) and 33 healthy volunteer, was 103 copies. K-ras mutations were found in 59.7%(40 out of 64) of DNAs of patients with PC, and 13.6% (3 out of 22) of CP. There is none mutation found in control samples. All patients with PC exhibited high levels of tumour marker CA 19-9, and the mutation types of K-ras is independence with the clinical characters. In conclusion, the one-step procedure discribed may be a useful clinical tool for analysing K-ras point mutations in blood samples. In addition, this method can be adapted for simultanous detection of multiple mutations and quantitation.