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

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

Abstract

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.

PMID:
30125495
PMCID:
PMC6157911
[Available on 2019-09-19]
DOI:
10.1021/jacs.8b06685

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