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Published July 28, 2014 | Accepted Version
Journal Article Open

Measuring Fate and Rate of Single-Molecule Competition of Amplification and Restriction Digestion, and Its Use for Rapid Genotyping Tested with Hepatitis C Viral RNA

Abstract

We experimentally monitored, at the single-molecule level, the competition among reverse transcription, exponential amplification (RT-LAMP), and linear degradation (restriction enzymes) starting with hepatitis C viral RNA molecules. We found significant heterogeneity in the rate of single-molecule amplification; introduction of the restriction enzymes affected both the rate and the "fate" (the binary outcome) of single-molecule amplification. While end-point digital measurements were primarily sensitive to changes in fate, the bulk real-time kinetic measurements were dominated by the rate of amplification of the earliest molecules, and were not sensitive to fate of the rest of the molecules. We show how this competition of reactions can be used for rapid HCV genotyping with either digital or bulk readout. This work advances our understanding of single-molecule dynamics in reaction networks and may help bring genotyping capabilities out of clinical labs and into limited-resource settings.

Additional Information

© 2014 Wiley-VCH Verlag GmbH & Co. Manuscript Received: 5 Mar 2014. Article first published online: 2 Jun 2014. Early View (Online Version of Record published before inclusion in an issue). This research was supported by NIH grants R01EB012946 and DP1OD003584, the Caltech Innovation Initiative, and NIH/NRSA training grant 5T32GM07616. We thank T. Schlappi for data analysis, A. Tucker-Schwartz for helpful discussions, and Whitney Robles for contributions to the preparation of this manuscript. R.F.I. has a financial interest in SlipChip Corp.

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