title: Fluorescence-based temperature control for polymerase chain reaction.
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. Emission 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 excitation 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 temperature measurements are accurate, no holding period is required even at rapid speeds. A custom instrument using fluorescence-based temperature monitoring with dynamic feedback control for temperature cycling amplified a fragment surrounding rs917118 from genomic DNA in 3min and 45s using 35 cycles, allowing subsequent genotyping by high-resolution melting analysis. Gold-standard thermocouple readings 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.
authors.affiliation: Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Lindsay N
authors.initials: LN
authors.lastname: Sanford
authors.affiliation: Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA. Electronic address: carl.wittwer@path.utah.edu.
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
mesh_terms: D004247:DNA / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D053803:Forensic Genetics; D015894:Genome, Human; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012235:Rhodamines / Q000737:chemistry; D044366:Transition Temperature
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; C022027:lissamine rhodamine B; D004247:DNA
keywords: Fluorescence; High-resolution melting; Polymerase chain reaction (PCR); Temperature monitoring