Proceedings of the National Academy of Sciences · 1999 · 1.7K citations · 46 references
MedicineGeneticsMolecular BiologyMolecular DiagnosticsMolecular OncologyOncogenesColorectal CancerMutation AnalysisOncogene MutationsPolymerase Chain ReactionRas Family OncogenesVariant DetectionAmplification-based Mutation DetectionDigital PcrFluorescent ProbesGenetic Mutation DetectionPolymerase Chain Reaction (Pcr) AssaysQuantitative Pcr DetectionSingle Nucleotide Variant AnalysisTumor Mutation Detection
The identification of predefined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. Here, we describe an approach for transforming the exponential, analog nature of the PCR into a linear, digital signal suitable for this purpose. Single molecules are isolated by dilution and individually amplified by PCR; each product is then analyzed separately for the presence of mutations by using fluorescent probes. The feasibility of the approach is demonstrated through the detection of a mutant ras oncogene in the stool of patients with colorectal cancer. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.
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Chris Heid, Junko Stevens, Kenneth J. Livak et al. · Genome Research · 1996 · 6.2K citations · Full text
Prevalence of ras gene mutations in human colorectal cancers
Johannes L. Bos, Eric R. Fearon, Stanley R. Hamilton et al. · Nature · 1987 · 1.9K citations