INVESTIGADORES
BELLI Carolina Barbara
artículos
Título:
Rapid detection of exon-1 NRAS activating mutations using universal heteroduplex generator technology
Autor/es:
BELLI, CAROLINA; DE BRASI, CARLOS; LARRIPA, IRENE
Revista:
HUMAN MUTATION
Editorial:
Wiley-Liss, Inc
Referencias:
Lugar: New York; Año: 2003 vol. 21 p. 132 - 137
ISSN:
1059-7794
Resumen:
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.