Rapid, Simple, and Accurate Detection of K-ras Mutations From Body Fluids Using Real-Time PCR and DNA Melting Curve Analysis

Abstract

The K-ras oncogene is one of the most useful genetic markers in screening for the presence of cancers because it is largely involved in tumorigenesis. However, most mutationdetection techniques are generally unsuitable for routine use, especially due to their timeconsuming, labor-intensive, and sensitive natures. Accordingly, we attempted to establish a new technique for analysis of K-ras alterations at codons 12 and 13 from body fluid specimens with tumor DNA using realtime polymerase chain reaction (PCR) with melting curve analysis (MCA). In this PCRMCA method, K-ras was easily genotyped by melting temperatures (Tm) that differ by 3.6°C to 11.6°C with an acceptable reproducibility of Tm. The shape of the melting curve gave easier interpretation. The detection sensitivity was at least 10 -3 . The entire process of the test was completed within 4 hours, saving 7 to 8 hours when compared to the current PCR-restriction fragment length polymorphism (RFLP) analysis. The examination of the MCA method using 38 clinical samples revealed the better detection rate (32% versus 24%) and diagnostic efficiency (100% versus 92%) compared with that of the PCR-RFLP. In conclusion, this small scale but high throughput method is acceptable for clinical use to analyze K-ras alterations from body fluids and plasma DNA, including small tumor DNA.