Ultraspecific and Amplification-Free Quantification of Mutant DNA by Single-Molecule Kinetic Fingerprinting

J Am Chem Soc. 2018 Sep 19;140(37):11755-11762. doi: 10.1021/jacs.8b06685. Epub 2018 Sep 5.


Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by 3 orders of magnitude, with a dynamic range of up to 5 orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug-resistance-conferring cancer mutation EGFR T790M (a single C → T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA / analysis*
  • DNA / genetics*
  • Drug Resistance, Neoplasm / genetics
  • ErbB Receptors / genetics
  • Fluorescent Dyes / chemistry
  • Humans
  • Kinetics
  • Microscopy, Fluorescence
  • Mutation
  • Polymerase Chain Reaction


  • Fluorescent Dyes
  • DNA
  • EGFR protein, human
  • ErbB Receptors