Asymmetric PCR-based strategy for genetic analysis of the p53 tumor suppressor gene in cell lines and tumor tissues.
A novel strategy for genetic analysis of the p53 tumor suppressor gene is described, based on direct sequencing of the asymmetric polymerase chain reaction (PCR) products. A set of 10 PCR primers was designed which allows to amplify and sequence highly conserved regions of the molecule, i.e. the target areas of p53 mutations. The stepwise optimization of RNA isolation, cDNA synthesis, PCR amplifications and sequencing resulted in a procedure which is faster and more reliable than the techniques used to search for p53 mutations so far. This and similar strategies should be applicable to the study of genetic alterations in antioncogenes or other classes of genes which suffer from subtle mutations potentially scattered along large segments of the molecule.
Bártek
J
J
Department of Immunology and Molecular Biology, Research Institute of Clinical and Experimental Oncology, Brno, Czechoslovakia.
Iggo
R
R
Vojtĕsek
B
B
Lane
D P
DP
D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification; D016147:Genes, Tumor Suppressor; D016158:Genes, p53; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000302:isolation & purification; D014407:Tumor Cells, Cultured / Q000378:metabolism
D009841:Oligonucleotides; D012313:RNA; D004247:DNA