Detection of single-base mutations in a mixed population of cells: a comparison of SSCP and direct sequencing.
Single-base-pair changes in several genes have been shown to be important in carcinogenesis. We have compared the sensitivity of two commonly used techniques for detection of single-base-pair changes. Defined populations of cells were prepared by mixing wild-type Chinese hamster ovary cells with cells carrying a known mutation in the CTP synthetase gene. RNA was extracted and analyzed for the mutation by single-strand conformational polymorphism and direct sequence analysis of polymerase chain reaction products. We found that both techniques were able to detect the mutation when it was present in 25% of the cells, but that direct sequencing was slightly more sensitive and able to detect the mutation when it was present in only 20% of the population.
Smith
T A
TA
Imperial Cancer Research Fund Department of Medical Oncology, St. Bartholomews Hospital, West Smithfield, London, England.
Whelan
J
J
Parry
P J
PJ
D000818:Animals; D016466:CHO Cells; D019731:Carbon-Nitrogen Ligases; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis / Q000379:methods*; D004277:DNA, Single-Stranded / Q000032:analysis*; D008025:Ligases / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity
D004277:DNA, Single-Stranded; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase