Peptide nucleic acid-mediated one-step PCR assay in detection of K-ras mutation
10.3724/SP.J.1008.2009.00762
- Author:
Han LIN
1
Author Information
1. Department of Gastroenterology
- Publication Type:Journal Article
- Keywords:
K-ras gene;
Pancreatic neoplasms;
Peptide nucleic acid;
Real-time quantitative PCR
- From:
Academic Journal of Second Military Medical University
2010;30(7):762-766
- CountryChina
- Language:Chinese
-
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 103 copies 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.
