Fluorescence-based temperature control for polymerase chain reaction q Lindsay N. Sanford a , Carl T. Wittwer b,⇑ a Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA b Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA article info Article history: Received 14 October 2013 Received in revised form 20 November 2013 Accepted 22 November 2013 Available online 28 November 2013 Keywords: Polymerase chain reaction (PCR) Fluorescence Temperature monitoring High-resolution melting abstract The ability to accurately monitor solution temperature is important for the polymerase chain reaction (PCR). Robust amplification during PCR is contingent on the solution reaching denaturation and annealing temperatures. By correlating temperature to the fluorescence of a passive dye, noninvasive monitoring of solution temperatures is possible. The temperature sensitivity of 22 fluorescent dyes was assessed. Emis- sion spectra were monitored and the change in fluorescence between 45 and 95 °C was quantified. Seven dyes decreased in intensity as the temperature increased, and 15 were variable depending on the excita- tion wavelength. Sulforhodamine B (monosodium salt) exhibited a fold change in fluorescence of 2.85. Faster PCR minimizes cycling times and improves turnaround time, throughput, and specificity. If tem- perature measurements are accurate, no holding period is required even at rapid speeds. A custom instru- ment using fluorescence-based temperature monitoring with dynamic feedback control for temperature cycling amplified a fragment surrounding rs917118 from genomic DNA in 3 min and 45 s using 35 cycles, allowing subsequent genotyping by high-resolution melting analysis. Gold-standard thermocouple read- ings and fluorescence-based temperature differences were 0.29 ± 0.17 and 0.96 ± 0.26 °C at annealing and denaturation, respectively. This new method for temperature cycling may allow faster speeds for PCR than currently considered possible. Ó 2013 Elsevier Inc. All rights reserved. The ability to accurately monitor and control solution tempera- ture during PCR 1 and melting analysis is crucial for successful ampli- fication and analysis of high-resolution melting curves. Due to practical limitations, temperature measurements are typically made externally to the sample solution. This produces significant solution- instrument temperature mismatches [1] which are exacerbated dur- ing rapid temperature transitions. One proposed solution for amelio- rating solution-instrument temperature discrepancies is to correlate the fluorescence of a passive dye (one that does not interact with DNA) to solution temperature for noninvasive monitoring in real time. This approach has been successfully demonstrated in harsh environment flow-field applications [2,3], to make temperature maps of microfluidic systems [4], and more recently, in commercial PCR instruments [1] to assess the differences in temperature recorded by the instrument and actual PCR solution temperatures. The ability to noninvasively monitor solution temperatures during PCR may provide more accurate sample temperatures and allow for faster cycling. Furthermore, the ability to dynamically control thermal cycling based on actual solution (and not external) temperature is enabled. The idea of utilizing fluorescence to con- trol PCR temperature cycling was first suggested in 1994 [5], and some progress has been made toward that goal. Fluorescence mon- itoring has been used to control extension times during PCR [6]. This allowed for heating (toward denaturation) to begin as soon as extension was complete, instead of waiting for a prespecified time interval to pass. However, this approach used a fluorescent dye that also interacts with DNA, potentially confounding the fluo- rescent signals for temperature measurement and real-time PCR monitoring. An alternative approach is to use a temperature-sensitive dye that does not interact with DNA (and that does not inhibit the PCR), but responds to changes in temperature with altered emis- sion intensity. For instance, the fluorescence of a temperature-sen- sitive dye has been used to adjust the power of a laser used to heat PCR mixtures from annealing to denaturation temperatures in a droplet-based PCR system [7] to demonstrate fluorescence-based heating control. However, complete thermal cycling control during PCR using fluorescence measurements requires the ability to con- trol all parts of PCR including heating, cooling, and holding times. We begin with an examination of 22 alternative dyes for fluo- rescence-based temperature monitoring, evaluating temperature 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.11.027 q Partial support for this research was provided by BioFire Diagnostics and Canon US Life Sciences. ⇑ Corresponding author. Address: Department of Pathology, University of Utah School of Medicine, 50 N. Medical Dr., Salt Lake City, UT 84132, USA. Fax: +1 801 581 6001. E-mail address: carl.wittwer@path.utah.edu (C.T. Wittwer). 1 Abbreviation used: PCR, polymerase chain reaction. Analytical Biochemistry 448 (2014) 75–81 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio