Rapid detection of K-ras mutations in bile by peptide nucleic acid-mediated PCR clamping and melting curve analysis: comparison with restriction fragment length polymorphism analysis.
BACKGROUND
Current methods for detection of K-ras gene mutations are time-consuming. We aimed to develop a one-step PCR technique using fluorescent hybridization probes and competing peptide nucleic acid oligomers to detect K-ras mutations in bile and to compare the efficacy with restriction fragment length polymorphism (RFLP) analysis.


METHODS
Bile samples were obtained from 116 patients with biliary obstruction, including gallstones (n = 64), benign biliary strictures (n = 6), pancreatic cancer (n = 20), and cholangiocarcinoma (n = 26). The DNA was extracted and subjected to K-ras mutation analysis by real-time PCR and RFLP analysis. Mutations were confirmed by direct sequencing. The sensitivity and specificity were calculated according to the clinical results.


RESULTS
The analysis time for real-time PCR was <1 h, whereas RFLP analysis took more than 2 days. With the sensor probe designed for the GAT (G12D) mutant in codon 12 of the K-ras gene, the real-time PCR method also detected the GTT (G12V) mutant. In contrast, a specific sensor probe for the TGT (G12C) mutant detected GAT (G12D), AGT (G12S), and GTT (G12V) mutants in addition to the TGT mutant. The real-time PCR assay allowed the detection of mutation in a 3000-fold excess of wild-type bile DNA. In bile, K-ras codon 12 mutations were detected in 16 of 46 malignant cases by real-time PCR with the TGT probe and 15 by RFLP analysis. All benign cases were wild type.


CONCLUSIONS
Real-time PCR with a cysteine-specific (TGT) sensor probe can rapidly detect K-ras gene mutations in bile and diagnose malignant biliary obstruction with high specificity.
Chen
Chiung-Yu
CY
Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Shiesh
Shu-Chu
SC
Wu
Sheu-Jen
SJ
D000368:Aged; D001646:Bile / Q000737:chemistry*; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics*; D018281:Cholangiocarcinoma / Q000175:diagnosis; D042883:Choledocholithiasis / Q000175:diagnosis; D003062:Codon; D005260:Female; D011905:Genes, ras; D006801:Humans; D020347:Lithiasis / Q000175:diagnosis; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000175:diagnosis; D020135:Peptide Nucleic Acids; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D018631:ras Proteins / Q000032:analysis / Q000235:genetics*
D014408:Biomarkers, Tumor; D003062:Codon; D020135:Peptide Nucleic Acids; D018631:ras Proteins