Allelic discrimination was performed with a panel of 80 different genomic DNAs (10 ng or 1 ng each in 5 μl reactions) using either the [A] Type-it Fast SNP Probe PCR Kit or [B] a genotyping master mix from Supplier A. Even when using low amounts of template, the Type-it Fast SNP Probe PCR Master Mix outperformed the master mix from Supplier A and provided tight clustering of alleles and reliable results. PCR was performed with a TaqMan® SNP genotyping assay for rs 951134 (aryl-hydrocarbon receptor repressor gene) and 2 no template controls (NTCs) and analyzed on an Applied Biosystems 7900HT instrument. Black: NTCs; blue: homozygous for T allele (FAM fluorescence); green: heterozygous samples; red: homozygous for A allele (VIC fluorescence). Data was analyzed with 98% confidence.
The Type-it Fast SNP Probe PCR Master Mix consists of highly specific HotStarTaq Plus Polymerase, which enables fast and easy reaction setup at room temperature and ensures highly specific amplification — even with low template amounts.
Buffer systems from other suppliers used for SNP genotyping often show poorer separation of allele clusters due to nonspecific binding of the mismatch probe. The Type-it Fast SNP Probe Buffer System provides increased discrimination by narrowing the probe melting temperature window, thereby reducing nonspecific binding of the mismatch probe. Wide and clear separation of allele clusters is obtained.
Average automated allele call rates for a panel of different DNAs were analyzed using 6 different TaqMan® MGB-based SNP genotyping assays and 1 ng of genomic DNA from each of the 60 samples. PCR was performed with the indicated products in 384-well plates in a 5 μl reaction volume. Allelic discrimination plate read was performed on an Applied Biosystems 7900HT instrument. The Type-it Fast SNP Probe PCR Kit consistently resulted in the highest call rates and the lowest error rates.
[A] The proprietary PCR buffer contains Q-Bond, a molecule that facilitates the reduction of the annealing step to 5 seconds. Q-Bond increases the affinity of Taq DNA polymerases for short single-stranded DNA fragments, reducing the time needed for successful primer annealing. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing the duration of the annealing step.