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


RESULTS
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.


CONCLUSIONS
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.
Tabone
Tania
T
Molecular Plant Breeding Co-operative Research Centre and School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA 5064, Australia. tania.tabone@ludwig.edu.au
Mather
Diane E
DE
Hayden
Matthew J
MJ
D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004171:Diploidy; D023381:Endpoint Determination; D018745:Genome, Plant / Q000235:genetics; D005838:Genotype; D001467:Hordeum / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors; D044366:Transition Temperature
D017931:DNA Primers