A method to detect ras point mutations in small subpopulations of cells.
Biochemical assays for ras mutations are capable of detecting a mutant allele only if it is present in at least 5% of cells tested. Further, ras mutation assays which utilize the polymerase chain reaction (PCR) are unable to distinguish a ras mutation in a small population of cells from mutations resulting from Taq DNA polymerase base misincorporation. We used a standard restriction fragment length polymorphism assay of PCR-amplified c-Ki-ras to detect codon 12 mutations in tumor cells and found a cumulative error frequency for Taq DNA polymerase of one codon 12 mutation per 2 X 10(4) molecules of total amplification product. The Taq polymerase-induced mutations were found to be multiple base transitions and represented a constant proportion of the amplification product at each step of the PCR. The ability to detect the in vitro generated mutation was dependent on the number of thermal cycles and the sensitivity of the detection assay. With these considerations in mind, we developed a two-step RFLP assay in which the thermal cycle number was kept low and molecules containing mutations at codon 12 were selectively amplified in the second step. We were able to detect a ras mutation occurring in 1 per 1000 cells (a two log improvement over standard RFLP methods) without detecting mutations resulting from Taq DNA polymerase infidelity.
Chen
J
J
Department of Medicine, State University of New York, Stony Brook 11794.
Viola
M V
MV
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D014407:Tumor Cells, Cultured
D003062:Codon; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase