High‐Throughput Genotyping Using the TaqMan Assay

AbstractThis unit describes the use of the 5‐nuclease allelic discrimination assay, or TaqMan, for genotyping with single nucleotide polymorphisms (SNPs).In this method, the region flanking the polymorphism, typically 100 to 150 bp, is amplified in the presence of two probes, each specific for one or the other allele. Probes have a fluor called “reporter” at the 5 end but do not fluoresce when free in solution because they have a “quencher“ at the 3 end that absorbs fluorescence from the reporter. The presence of two probes allows one to detect both alleles in a single tube. Moreover, because probes are included in the PCR, genotypes are determined without any post‐PCR processing, a feature that is unavailable with most other genotyping methods. The design of probes and primers is described first, followed by PCR conditions, and finally methods for assigning genotypes in an automated fashion are discussed.