Digital PCR

Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41. doi: 10.1073/pnas.96.16.9236.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Base Sequence
  • Colorectal Neoplasms / genetics*
  • DNA Primers
  • Fluorescent Dyes
  • Genes, ras*
  • Humans
  • Point Mutation*
  • Polymerase Chain Reaction / instrumentation
  • Polymerase Chain Reaction / methods*
  • Proto-Oncogene Proteins p21(ras) / chemistry
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Spectrometry, Fluorescence / instrumentation
  • Spectrometry, Fluorescence / methods
  • Taq Polymerase
  • Transcription, Genetic

Substances

  • DNA Primers
  • Fluorescent Dyes
  • Taq Polymerase
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)