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Open Access Highly Accessed Open Badges Methodology article

Temperature Switch PCR (TSP): Robust assay design for reliable amplification and genotyping of SNPs

Tania Tabone12, Diane E Mather1 and Matthew J Hayden13*

Author Affiliations

1 Molecular Plant Breeding Co-operative Research Centre and School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA 5064, Australia

2 Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, VIC 3050, Australia

3 Department of Primary Industries Victoria, Victorian AgriBiosciences Centre, La Trobe R&D Park, Bundoora, VIC 3083, Australia

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BMC Genomics 2009, 10:580  doi:10.1186/1471-2164-10-580

Published: 3 December 2009



Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs). Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP), a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays.


We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method.


Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.