PCR-based approaches for detection of mutated ras genes.

PAP: Detection of ultra rare mutations depends on P* oligonucleotides: ?Sleeping Beauties? awakened by the kiss of pyrophosphorolysis

“…1) [Parsons and Heflich, 1997b]. Three types of methods for direct analysis of point mutations exist: 1) preferential amplification of the rare allele (e.g., PCR amplification of specific alleles [PASA] [Sarkar et al, 1990], mutagenically separated PCR [MS-PCR] [Rust et al, 1993], allele-specific competitive blocker PCR [ACB-PCR] [Orou et al, 1995], mismatch amplification mutation assay [MAMA] [Cha et al, 1992], blocker-PCR [Seyama et al, 1992], peptide nucleic acid [PNA] clamping blocker PCR [Thiede et al, 1996], ligation chain reaction [LCR] [Wiedmann et al, 1994], and Gap-LCR [Abravaya et al, 1995]); 2) preferential destruction of the wild-type allele (e.g., RFLP/PCR [Felley-Bosco et al, 1991], PCR/RFLP [van Mansfeld and Bos, 1992], and mismatch protection from exonuclease cleavage by MutS followed by PCR [MutEx/ PCR] [Parsons and Heflich, 1997a]); or 3) spatial separation of the mutant and wild-type alleles (e.g., single-strand conformation polymorphism [SSCP] [Orita et al, 1989], denaturing gradient gel electrophoresis [DGGE] [Fischer and Lerman, 1983], constant denaturant capillary electrophoresis [CDCE] [Khrapko et al, 1994], multiple rounds of CDCE [Khrapko et al, 1997], competitive mobility shift assay [CMSA] [Chen et al, 1996], and magnetic mismatch binding beads [M 2 B 2 ] [Genecheck, Fort Collins, CO]). PASA, also called AS-PCR or ARMS (a widely used method), routinely detects a mutated allele in 40-200 copies of the wild-type allele [Sarkar et al, 1990].…”

“…RFLP/PCR, the most sensitive of the methods, has been reported to detect a mutated allele in 10 8 wild-type alleles [Felley-Bosco et al, 1991], but 1) the method is highly labor intensive, 2) the mutation must be within an appropriate restriction endonuclease site, and 3) the cleavage efficiencies of many restriction endonucleases limit the selectivity of the method to a range of 1:10 3 to 1:10 5 [Parsons and Heflich, 1997b]. PCR/RFLP and its many variants, such as quantitative enriched PCR [Ronai and Minamoto, 1997], inverse restriction site mutation analysis [Jenkins et al, 1999], modified nucleotide analogs and buffers [Day et al, 1999], and modification using ligation [Kaur et al, 2002], achieved a selectivity of 1:10 6 [van Mansfeld and Bos, 1992]. ACB-PCR detected a mutated allele in 10 5 wild-type alleles [Parsons and Heflich, 1998a] and its combination with MutEX enrichment increased the selectivity to 1:10 7 [Parsons and Heflich, 1998b].…”

Efeitos tardios dos praguicidas organoclorados no homem

“…As conseqüências de tal decréscimo causado pelo heptacloro, seja por supressão na taxa de transcrição ou por degradação de RNAm, não são conhecidas. De qualquer modo, esse achado é interessante, dado que o gene NRAS faz parte de uma família de protoncogenes que estão envolvidos no desenvolvimento e progressão de diferentes tipos de neoplasias 68 . Do ponto de vista epidemiológico, a avaliação do potencial carcinogênico dos organoclorados e dos demais praguicidas é extremamente complexa.…”

Detection of hotspot mutations and polymorphisms using an enhanced PCR-RFLP approach

“…Those with greater sensitivity are necessarily more complicated in terms of methodology and, sometimes, less robust and reproducible. Ethidium gelbased PCR-RFLP is widely used and is perhaps, the simplest method for detection of known mutations in cancer-related genes, and for genotyping a wide range of other human diseases [Bazrafshani et al, 2000;Bos and Van Mansfeld, 1992;Eiken et al, 1991;Friedman et al, 1990;Parsons and Heflich, 1997;Plendl et al, 2001;van Mansfeld and Bos, 1992]. A drawback for the application of this method in the field of cancer, however, is that it cannot detect mutant alleles present in less than about 5-10% of wild-type alleles [Haliassos et al, 1989].…”