Angelika Heißl, Barbara Arbeithuber, Irene Tiemann-Boege,
"High-Throughput Genotyping with TaqMan Allelic Discrimination and Allele-Specific Genotyping Assays"
, in White, Stefan J., Cantsilieris, Stuart (Eds.): Genotyping. Methods and Protocols, Serie Methods in Molecular Biology, Vol. 1492, Springer Protocols, New York, Seite(n) 29?57, 2017, ISBN: 978-1-4939-6442-0
High-Throughput Genotyping with TaqMan Allelic Discrimination and Allele-Specific Genotyping Assays
Sprache des Titels:
Genotyping. Methods and Protocols
Real-time PCR based genotyping methods such as TaqMan allelic discrimination assays and allele-specific genotyping are powerful tools for high-throughput genotyping of single nucleotide polymorphisms and short tandem repeats. TaqMan genotyping assays use two dual-labelled probes that emit a fluorescent signal specific to the allele of the target within one reaction. In comparison, allele-specific genotyping relies on the differential amplification of the alleles by allele-specific primer pairs contained in two different reactions and detected with an intercalating dye. Both assays are particularly useful when screening a handful of polymorphisms in hundreds of samples, either derived from different individuals, different tissues, or even different pre-amplified single DNA molecules.
Although real-time PCR based methods such as TaqMan are well-established, alternative methods, like allele-specific genotyping, are powerful alternatives, especially for genotyping short tandem repeat length polymorphisms. In this chapter, we describe all relevant aspects when developing an assay for a new locus, using either TaqMan or allele-specific genotyping, such as primer and probe design, optimization of reaction conditions, the experimental procedure used for typing hundreds of samples, and finally evaluation of the data. Our goal is to provide a guideline for developing genotyping assays using these two approaches that render reliable and reproducible genotype calls with minimal optimization.