Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

• Published in: Bratislavské lékarské listy Vol. 113; no. 2; pp. 73 - 79
• Main Authors: Ugorcakova, J, Hlavaty, T, Novotna, T, Bukovska, G
• Format: Journal Article
• Language: English
• Published: Slovakia 2012
• Subjects: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor - genetics; Digestive System Neoplasms - diagnosis; Digestive System Neoplasms - genetics; Female; Genotyping Techniques; Humans; Male; Middle Aged; Point Mutation; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins p21(ras); ras Proteins - genetics; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA
• ISSN: 0006-9248
• DOI: 10.4149/BLL_2012_018

The determination of gene mutations is important for the diagnosis and prognosis of various gastrointestinal cancers. The aim of our study was to develop a new procedure for the analysis of KRAS gene mutation by application of the real-time PCR method. The detection process requires discriminate trace amount of mutant allele in a large excess of wild-type DNA in various samples. The real-time PCR based technique using hybridization probes for five most frequently KRAS codon 12 mutations and WT specific peptide nucleic acid (PNA) was performed. Our multiplex detection system was tested in various DNA samples (tissue, bile, pancreatic juice) of patients with different diagnoses of gastrointestinal tract disease obtained by endoscopy and ERCP. We designed and optimized the real-time PCR conditions and tested various amount of PNA in PCR reaction to suppress amplification of the wild-type DNA. We determined the interassay variability of the melting temperatures and the results of mutation testing were confirmed by DNA sequencing with the 100 % accuracy. Incidence of searched mutations was 67.5 % in cohort of 40 patients; for KRASG12D it was in 44.4 %, KRASG12V in 22.2 %, KRASG12S in 14.8 %, KRASG12A in 14.8 % and KRASG12C in 3.8 %. The sensitivity of the assays is 1x10-5. Advantages of this technique are rapidity, accuracy and it is generally easy to perform. This method can be adapted for synchronic detection of multiple mutations and after readjustment by other type mutation of KRAS gene may serve as useful clinical tool for analyzing point mutations in various clinical samples (Tab. 3, Fig. 3, Ref. 42).