Quantification of random genomic mutations.
Cancer cells contain numerous clonal mutations. It has been theorized that malignant cells sustain an elevated mutation rate and, as a consequence, harbor yet larger numbers of random point mutations. Testing this hypothesis has been precluded by lack of an assay to measure random mutations-that is, mutations that occur in only one or a few cells of a population. We have established a method that has permitted us to detect and identify rare random mutations in human cells, at a frequency of 1 per 10(8) base pairs. The assay is based on gene capture, by hybridization with a uracil-containing probe, followed by magnetic separation. Mutations that render the mutational target sequence non-cleavable by a restriction enzyme are quantified by dilution to single molecules and real-time quantitative PCR amplification. The assay can be extended to quantify mutation in any DNA-based organism, at different sites in the genome, in introns and exons, in unselected and selected genes, and in proliferating and quiescent cells.
Bielas
Jason H
JH
The Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington School of Medicine, Department of Pathology, 1959 N.E. Pacific Street, Seattle, Washington 98195-7705, USA.
Loeb
Lawrence A
LA
D002874:Chromosome Mapping / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D005347:Fibroblasts; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008280:Magnetics; D008957:Models, Genetic; D015233:Models, Statistical; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*