High-Throughput Genotyping with TaqMan Allelic Discrimination and Allele-Specific Genotyping Assays.
Real-time PCR-based genotyping methods, such as TaqMan allelic discrimination assays and allele-specific genotyping, are particularly useful when screening a handful of single nucleotide polymorphisms in hundreds of samples; either derived from different individuals, tissues, or pre-amplified DNA. 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 (STR) length polymorphisms. Here, we describe all relevant aspects when developing an assay for a new SNP or STR using either TaqMan or allele-specific genotyping, respectively, such as primer and probe design, optimization of reaction conditions, the experimental procedure for typing hundreds of samples, and finally the data evaluation. Our goal is to provide a guideline for developing genotyping assays using these two approaches that render reliable and reproducible genotype calls involving minimal optimization.
Institute of Biophysics, Johannes Kepler University, Gruberstraße 40, 4020, Linz, Austria.
Angelika
A
Heissl
Institute of Biophysics, Johannes Kepler University, Gruberstraße 40, 4020, Linz, Austria.
Barbara
B
Arbeithuber
Institute of Biophysics, Johannes Kepler University, Gruberstraße 40, 4020, Linz, Austria. Irene.tiemann@jku.at.
Irene
I
Tiemann-Boege
D000483:Alleles; D018076:DNA, Complementary / Q000235:genetics; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats; D020641:Polymorphism, Single Nucleotide; D012313:RNA / Q000235:genetics*; D015347:RNA Probes; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary; D015347:RNA Probes; D012313:RNA
5′ endonuclease assay; Allele-specific genotyping; Allelic discrimination; Dual-labeled probes; Microsatellites; Real-time PCR; SNP genotyping; SYBR Green I method; Short tandem repeats; TaqMan assay