Single-strand conformation polymorphism analysis to detect p53 mutations: characterization and development of controls.
Single-strand conformation polymorphism (SSCP) analysis is widely used to prescreen mutations in p53 gene. However, standardization of SSCP to detect p53 mutations has rarely been pursued so far. We have developed complete conditions for a temperature-controlled nonradioactive SSCP for mutation detection in amplified p53 exons 4-8, where mutations frequently occur in human tumors. Easily obtainable and clearly distinguishable positive controls were developed by replacing the regular 5' primers in amplification with primers that include one to three mutated sites. Careful purification of the amplified products by gel electrophoresis appeared to be essential. The efficiency of the method was studied by using previously sequenced samples with p53 mutations and the various positive controls. The use of two temperatures (exon 4: 4 degrees C and 15 degrees C; other exons: 4 degrees C and 20 degrees C) in combination with other optimized conditions resulted in 98% efficiency in mutation detection, which was considered sufficient for routine screening.
Welsh
J A
JA
Laboratory of Human Carcinogenesis, NCI, National Institutes of Health, Bethesda, MD 20892, USA. welshj@intra.nci.nih.gov
Castrén
K
K
Vähäkangas
K H
KH
D005091:Exons; D016158:Genes, p53; D006801:Humans; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D012015:Reference Standards; D015203:Reproducibility of Results; D013696:Temperature