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

Abstract

Abstract:Objectives: 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. Background: The detection process requires discriminate trace amount of mutant allele in a large excess of wild-type DNA in various samples. Methods:The real-time PCR based technique using hybridization probes for fi ve most frequently KRAS codon 12 mutations and WT specifi c 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. Results: We designed and optimized the real-time PCR conditions and tested various amount of PNA in PCR reaction to suppress amplifi cation of the wild-type DNA. We determined the interassay variability of the melting temperatures and the results of mutation testing were confi rmed by DNA sequencing with the 100 % accuracy. Incidence of searched mutations was 67.5 % in cohort of 40 patients; for KRAS G12D it was in 44.4 %, KRAS G12V in 22.2 %, KRAS G12S in 14.8 %, KRAS G12A in 14.8 % and KRAS G12C in 3.8 %. The sensitivity of the assays is 1x10 -5 . Conclusions: 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). Full Text in PDF www.elis.sk.

The role of miRNAs targeting K-ras and APC genes in colorectal cancer

“…K-ras is one of the recommended biomarkers for CRC (29 higher in CRC tumor tissues compared to normal tissues, its mRNA level was altered irregularly (30). In our study, when K-ras expression levels were examined between tumor tissue and non-tumor adjacent tissue samples of the patients, we observed higher K-ras levels in tumor tissues.…”

Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells

“…Lytic enzymes are diverse in respect to substrate specificity. CD11 and CDG amidases of Peptoclostridium difficile are highly active against C. difficile clinical isolates while ineffective against Bacillus or Staphylococcal species [48]. Substrate specificity of PlyCP39O and PlyCP26F endolysins from clostridial phages phiCP39O and phiCP26F, respectively, is very narrow and includes only C. perfringens strains, as these enzymes did not lyse non-perfringens clostridial isolates.…”

“…Here, we experimentally confirmed the dependence of LysB lytic activity on the presence of zinc ions (Table 1). Amidase_2 domain with Zn 2+ in the catalytic center is also present at the N-terminal part of CP25L endolysin of C. perfringens phage vB_CpeS-CP51 [17], three predicted endolysins from podoviruses of C. perfringens, ΦCPV4, ΦZP2, and ΦCP7R [47], and a putative lytic enzyme named CDG from Peptoclostridium difficile DA00211 strain [48]. Further analysis showed low LysB amino acid sequence identity (31.67%) to the primary sequence of Thermus scotoductus phage Ts2631 endolysin (GenBank accession no.…”