Digital PCR.
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.
Vogelstein
B
B
The Howard Hughes Medical Institute and the Johns Hopkins Oncology Center, Baltimore, MD 21231, USA. vogelbe@welchlink.welch.jhu.edu
Kinzler
K W
KW
D019943:Amino Acid Substitution; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000235:genetics*; D017931:DNA Primers; D005456:Fluorescent Dyes; D011905:Genes, ras; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D019914:Taq Polymerase; D014158:Transcription, Genetic
D017931:DNA Primers; D005456:Fluorescent Dyes; D019914:Taq Polymerase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)