Sensitive detection of p53 gene mutations by a 'mutant enriched' PCR- SSCP technique
- 1 March 1998
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 26 (5) , 1356-1358
- https://doi.org/10.1093/nar/26.5.1356
Abstract
For the rapid and sensitive detection of p53 'hot spot' mutations, we combined polymerase chain reaction based single-strand conformational polymorphism (PCR-SSCP) analysis with sequence specific-clamping by peptide nucleic acids (PNAs) in a one-step reaction tube protocol. For this purpose, we designed two PNA molecules comprising aa 246-250 of exon 7 and aa 270-275 of exon 8, respectively, to suppress the amplification of wild-type p53 allelic variants during PCR amplification. Using this method in a survey of 20 brush cytology samples from lung cancer patients, we were able to detect five p53 point mutations occurring in codons 248, 249 and 273 which could not be retrieved by conventional PCR-SSCP. Thus, allelic suppression by PNA molecules opens a way to largely improve the sensitivity of existing PCR-SSCP protocols (approximately 10-50-fold) and could be useful in the detection of 'hot spot' oncogene lesions in histological samples containing only a small number of cancer cells.Keywords
This publication has 11 references indexed in Scilit:
- Nested genetic bit analysis (N-GBA) for mutation detection in the p53 tumor suppressor gene.Nucleic Acids Research, 1997
- Potassium permanganate and tetraethylammonium chloride are a safe and effective substitute for osmium tetroxide in solid-phase fluorescent chemical cleavage of mismatchNucleic Acids Research, 1997
- CCR: a rapid and simple approach for mutation detectionNucleic Acids Research, 1997
- Point Mutations of the Mxil Gene are Rare in Prostate CancersThe Prostate, 1996
- Nonradioactive Characterization of Low-Level Heteroplasmic Mitochondrial DNA Mutations by SSCP-PCR EnrichmentBioTechniques, 1996
- Modified oligonucleotides as therapeutic and diagnostic agentsCurrent Opinion in Biotechnology, 1995
- Peptide nucleic acids and their potential applications in biotechnologyTrends in Biotechnology, 1993
- Single base pair mutation analysis by PNA directed PCR clampingNucleic Acids Research, 1993
- Sensitive Detection of p53 Gene Mutations in Esophageal Endoscopic Biopsy Specimens by Cell Sorting Combined with Polymerase Chain Reaction Single‐strand Conformation Polymorphism AnalysisJapanese Journal of Cancer Research, 1992
- Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reactionGenomics, 1989