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SELECTBIO Conferences Liquid Biopsies 2019


Toward Perfect Detection of Un-amplified Biomarkers with Single-Molecule Kinetic Fingerprinting

Alexander Johnson-Buck, Research Assistant Professor, University of Michigan

Conventional techniques for detecting rare DNA and RNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. In addition, some classes of biomarkers, such as epigenetic modifications and proteins, cannot be amplified directly. While amplification-free methods exist, they rarely achieve single-molecule sensitivity; furthermore, even when such sensitivity is achieved, the ability of most methods to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics and/or nonspecific binding of probes to assay surfaces. We show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by three orders of magnitude.  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 the single-base selectivity of 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. Using super-resolution analysis methods, the dynamic range of the technique is increased to approximately five orders of magnitude, comparable to that of droplet digital PCR. We also explore methods of increasing sensitivity in surface binding assays through rational engineering of mass transfer to the imaging surface, and in some cases achieve detection limits in the low attomolar range, an improvement of >50-fold relative to passive diffusion alone. These results suggest the utility of single-molecule kinetic measurements for the direct, digital detection of nucleic acids and other analytes with extremely high specificity.

Add to Calendar ▼2019-03-27 00:00:002019-03-29 00:00:00Europe/LondonLiquid Biopsies 2019Liquid Biopsies 2019 in Coronado Island, CaliforniaCoronado Island,