期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 140, 期 37, 页码 11755-11762出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.8b06685
关键词
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资金
- Michigan Economic Development Corporation MTRAC for Life Sciences
- University of Michigan MCubed 2.0 program
- James Selleck Bower Permanently Endowed Innovative Promise Funds for Cancer Research of the University of Michigan Rogel Cancer Center
- Fast Forward GI Innovation Fund
- NIH [R21 CA204560]
- NSF MRI-R2-ID [DBI-0959823]
- NATIONAL CANCER INSTITUTE [R21CA204560] Funding Source: NIH RePORTER
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 S 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.
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