Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction

von Kanel, Thomas; Gerber, Dominik; Wittwer, Carl T; Hermann, Mark; Gallati, Sabina (2011). Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction. Analytical biochemistry, 419(2), pp. 161-7. San Diego, Calif.: Elsevier 10.1016/j.ab.2011.08.041

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Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.

Item Type:	Journal Article (Original Article)
Division/Institute:	04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Paediatric Medicine
UniBE Contributor:	Gallati, Sabina
ISSN:	0003-2697
Publisher:	Elsevier
Language:	English
Submitter:	Anette van Dorland
Date Deposited:	04 Oct 2013 14:24
Last Modified:	05 Dec 2022 14:07
Publisher DOI:	10.1016/j.ab.2011.08.041
PubMed ID:	21925477
Web of Science ID:	000296119400014
URI:	https://boris.unibe.ch/id/eprint/8343 (FactScience: 213869)