Allelic Discrimination

TaqMan SNP Genotyping Assays provide optimized assays for genotyping single nucleotide polymorphisms (SNPs). These products use the 5' nuclease assay for amplifying and detecting specific SNP alleles in purified genomic DNA samples. Each assay allows researchers to genotype individuals for a specific SNP.

Assay Components

Each SNP Genotyping Assay Mix contains:

  • Sequence-specific forward and reverse primers to amplify the SNP of interest
  • Two TaqMan MGB probes: one probe labeled with VIC dye detects Allele 1 sequence, one labeled with FAM dye detects Allele 2 sequence.
  • A minor groove binder (MGB). This modification increases the melting temperature without increasing probe length which allows for shorter probes. This results in greater differences in melting temperature values between matched and mismatched probes, with produces more allelic discrimination.
  • A nonfluorescent quencer (NFQ) at the 3' end of the probe. Because the quencher does not fluoresce, ABI sequence detections systems can measure reporter dye contributions more accurately.

5' Nuclease Assay

During PCR, the following events occur:

  1. Each TaqMan MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Forster-type energy transfer.
  2. AmpliTaq Gold DNA polymerase cleaves only probes that are hybridized to the target. Mismatches between a probe and target reduce the efficiency of probe hybridization. Furthermore, the enzyme is more likely to displace a mismatched probe without cleaving it, which does not produce a fluorescent signal.
  3. Cleavage separates the reporter dye from the quencher dye, which results in increased fluorescence by the reporter. The increase in fluorescence signal occurs only if the amplified target sequence is complementary to the probe. Thus, the fluorescence signal generated by PCR amplification indicates which alleles are present in the sample.