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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 5, Iss. 3 — Mar. 1, 2014
  • pp: 737–751

Microdroplet temperature calibration via thermal dissociation of quenched DNA oligomers

Eric W. Hall and Gregory W. Faris  »View Author Affiliations

Biomedical Optics Express, Vol. 5, Issue 3, pp. 737-751 (2014)

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The development of microscale analytical techniques has created an increasing demand for reliable and accurate heating at the microscale. Here, we present a novel method for calibrating the temperature of microdroplets using quenched, fluorescently labeled DNA oligomers. Upon melting, the 3′ fluorophore of the reporter oligomer separates from the 5′ quencher of its reverse complement, creating a fluorescent signal recorded as a melting curve. The melting temperature for a given oligomer is determined with a conventional quantitative polymerase chain reaction (qPCR) instrument and used to calibrate the temperature within a microdroplet, with identical buffer concentrations, heated with an infrared laser. Since significant premelt fluorescence prevents the use of a conventional (single-term) sigmoid or logistic function to describe the melting curve, we present a three-term sigmoid model that provides a very good match to the asymmetric fluorescence melting curve with premelting. Using mixtures of three oligomers of different lengths, we fit multiple three-term sigmoids to obtain precise comparison of the microscale and macroscale fluorescence melting curves using “extrapolated two-state” melting temperatures.

© 2014 Optical Society of America

OCIS Codes
(120.6780) Instrumentation, measurement, and metrology : Temperature
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.3890) Medical optics and biotechnology : Medical optics instrumentation

ToC Category:
Biosensors and Molecular Diagnostics

Original Manuscript: October 9, 2013
Revised Manuscript: February 4, 2014
Manuscript Accepted: February 7, 2014
Published: February 13, 2014

Eric W. Hall and Gregory W. Faris, "Microdroplet temperature calibration via thermal dissociation of quenched DNA oligomers," Biomed. Opt. Express 5, 737-751 (2014)

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