Received on July 16, 2003
Accepted on December 16, 2003

Molecular Diagnostics and Genetics

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

¹ Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
² Department of Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
³ Clinical Biochemistry Research Laboratory, Veterans General Hospital, Taipei, Taiwan

^* To whom correspondence should be addressed. E-mail: hsieh{at}mail.ncku.edu.tw.

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 gallstone (n = 64), benign biliary stricture (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.

Conclusion: 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.