Rapid detection of exon 1 NRAS gene mutations using universal heteroduplex generator technology.
Specific NRAS oncogene missense mutations have been frequently found in some tumors and several hematological diseases, especially in those of myeloid origin. There is a wide range of PCR-based methods for screening and detection of NRAS exon 1 single-base substitutions. However, there are disadvantages and ambiguities associated with these techniques because all of them require either separate probes, separate PCR amplifications, or complicated post-PCR manipulations. This report describes a new approach for detection of NRAS gene mutations at codon 12 and 13 based on the DNA heteroduplex analysis method. The strategy relies upon differential electrophoretic behavior of induced heteroduplex molecules formed by cross-hybridization of two PCR-amplified species, the sample under analysis and the synthetic universal heteroduplex generator (UHG). The screening of a panel of all codon 12 and 13 NRAS mutant DNA variants indicated that this approach discriminates all 12 relevant mutations. The sensitivity of the method was estimated by a competitive assay where mutant alleles could be detected at a dilution level of 1 to 16 wild-type alleles. This UHG technology was tested on some clinical samples previously studied by PCR-ASO. This methodology is highly specific, sensitive, and achieves an appreciable reduction in workload and time because it requires one PCR amplification followed by polyacrylamide gel electrophoresis in standard conditions. We propose that this new approach may be applied as an alternative strategy for codon 12-13 NRAS mutations and it could be easily incorporated into the range of routine assays performed in oncology laboratories.
Belli
Carolina
C
Departamento de Genética, Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires, Argentina. cbelli@hematologia.anm.edu.ar
De Brasi
Carlos
C
Larripa
Irene
I
D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics; D005091:Exons / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D020180:Heteroduplex Analysis / Q000379:methods; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009190:Myelodysplastic Syndromes / Q000235:genetics; D009447:Neuroblastoma / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured; D015678:Viral Structural Proteins / Q000235:genetics
D003062:Codon; D004273:DNA, Neoplasm; D009692:Nucleic Acid Heteroduplexes; D015678:Viral Structural Proteins; D004247:DNA; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)