SNP Genotyping Assays

• Minor groove binder (MGB) technology enables TaqMan probes to discriminate between highly homologous allele sequences
• Robust design pipeline with 90+ parameters for optimal primer-probe combinations
• Functional quality testing with 20 gDNA samples for each human assay
• Flexible formats, including single tubes; 48-, 96-, and 384-well plates; and OpenArray plates, to accommodate various throughput and number of targets and samples
• Backed by more than 296,000 publications and over 300 patents
• TaqMan Performance Guarantee (terms and conditions apply) provides confidence that assays will work as described

Each TaqMan SNP Genotyping assay includes two differentially labeled, allele-specific TaqMan MGB probes and a PCR primer pair that uniquely amplify and offer outstanding analytical specificity for the allele of interest. Choose an assay from one of more than 17 million predesigned assays or customize your own using our Custom Assay Design Tool.

Like all TaqMan Assays, TaqMan SNP genotyping assays require a double-stranded DNA template, the Taq polymerase enzyme, and two primers—forward and reverse—specific to the sequence to be amplified. Unlike gene expression qPCR, SNP detection requires two probes with different fluorescent reporters to differentiate homozygous from heterozygous samples.

The first probe, labeled with VIC fluorescent dye, detects the first allele sequence, while the second probe, labeled with FAM fluorescent dye, detects the second allele sequence. If the sample is homozygous for allele 1, the fluorescence readout or 'allelic discrimination plot' will show mostly VIC fluorescence. If the sample is homozygous for allele 2, the plot will mainly show signal from the FAM dye. If the sample is heterozygous, there should be roughly equal signal for each dye. Across an entire plate, the data should resolve into three discrete clusters. Exceptions can occur if the minor allele frequency is very low, if there is a copy number variation (CNV) of the gene in question, or if it lies on the X chromosome.

For their TaqMan SNP genotyping assays, the context sequence of the assay is always listed in the forward orientation, regardless of the strand on which the SNP is typically reported. It is important to compare the context sequence of the SNP assay to the SNP sequence in the NCBI’s dbSNP database to determine which dye is associated with the minor allele.

Discrimination of a single base

Three key factors contribute to the discrimination of a single base mismatch in genotyping assays:

1. Disruptive effect on hybridization: A mismatched probe has a lower melting temperature (T_m) than a perfectly matched probe. During PCR, selecting the appropriate annealing/extension temperature favors the hybridization of exact-match probes over mismatched ones.
2. Competitive binding: The assay is conducted with both probes present in the same reaction tube. Exact-match probes bind more stably, preventing mismatched probes from binding.
3. Probe displacement and cleavage: The 5' end of the probe must start to be displaced before cleavage by the 5’ nuclease activity of Taq DNA polymerase, which recognizes a forked structure with a displaced 5’ strand of at least 1 to 3 nucleotides. Mismatched probes dissociate faster than exact-match probes, allowing less time for cleavage and promoting dissociation over cleavage.