TaqMan® Genotyping Master Mix Real-time PCR master mix tailored for SNP genotyping studies Tailored for unrivaled cluster resolution for unambiguous SNP allelic discrimination. Optimized for ) genotyping applications, including: ™ AM e Y (F • Candidate gene studies el All • Drug target validation • Disease association studies • Population genetics ® • Linkage mapping Allele X (VIC ) • Agricultural applications • Copy number variation analysis Introduction Benefits Optimized formulation for exceptional TaqMan® Genotyping Master Mix is • Specifically formulated for endpoint performance designed to deliver reliable, cost- fluorescent detection of SNPs and TaqMan® Genotyping Master Mix is a effective SNP detection for accurate and insertions/deletions convenient 2X mix for TaqMan® probe- reproducible allelic discrimination. The • Discrete clusters and high call rates based genotyping reactions. It includes master mix optimizes the preferential for accurate and reproducible allelic the following components: binding of the allele-specific probe, discrimination providing exceptional separation and • Reliable discrimination of SNPs in • AmpliTaq Gold® DNA Polymerase, UP clustering of alleles and consistently difficult targets (Ultra Pure), a highly purified DNA strong fluorescent signals. Powered • Excellent room temperature stability polymerase. This hot-start enzyme with the highly purified AmpliTaq Gold® for flexible pre- and post-PCR setup is inactive at room temperature, DNA Polymerase, UP (Ultra Pure), and analysis so reactions can be set up on the TaqMan® Genotyping Master Mix can • Universal thermal cycling conditions benchtop. The enzyme is activated replace TaqMan® Universal PCR Master for consistent results during thermal cycling Mix in existing SNP genotyping proto- • Validated for use with TaqMan® • Optimized components including cols using the same reaction setup and SNP Genotyping Assays, TaqMan® buffer and dNTPs for consistent, thermal cycling conditions. Copy Number Assays, and TaqMan® reliable genotypes Mutation Detection Assays • Passive internal reference based on proprietary ROX™ dye for precise data analysis Setting a new standard for allelic secondary structure. Human gDNA discrimination samples were genotyped for a SNP in 6.0 For clear genotyping results, each a GC-rich region using a TaqMan® SNP allele-specific TaqMan® probe must Assay to genotype dbSNP rs12214 in the 5.0 yield bright and consistent fluorescent cathepsin D gene. As shown in Figure 3, signals to provide discrete clusters TaqMan® Genotyping Master Mix yields 4.0 that are widely separated, indicating brighter fluorescent signals, tighter AM™) excellent specificity. The performance clusters, and more accurate allele call- ® 3.0 of TaqMan Genotyping Master Mix ing compared to a mix from vendor “Si”. Allele Y (F was tested using 3 ng samples of These data demonstrate that TaqMan® 2.0 human genomic DNA (gDNA) and a Genotyping Master Mix provides higher validated SNP assay to genotype dbSNP call rates for reliable SNP genotyping in rs2293052 in the gene NOS1. The result- difficult targets, eliminating the need to 1.0 ing cluster plot [Figure 1] shows strong retest uncalled samples. fluorescent signals for each allele and 0.0 clear separation between the three Copy number variation applications 0.3 0.8 1.3 1.8 2.3 2.8 Copy number variation is an important Allele X (VIC®) clusters—easily discriminating the two homozygous and one heterozygous polymorphism in the human genome Figure 1. TaqMan® Genotyping Master Mix genotypes. In a comparison against that can be associated with certain provides bright fluorescence signals for genomic disorders as well as some discrete, well-separated allelic clusters. five commercially available mixes, TaqMan® Genotyping Master Mix shows simple genetic and complex diseases. Cluster plot of 94 gDNA samples and two ® no-template controls (NTCs) genotyped the highest average call rate (Figure 2). TaqMan Genotyping Master Mix, ® ® using TaqMan SNP Genotyping Assay Tight, well-separated clusters for each used with TaqMan Copy Number C__15969983_10, with PCR performed on genotype provide exceptional call rates Assays, provides relative quantitation the GeneAmp® PCR System 9700 and allelic and, most importantly, accurate and of an experimental gene compared discrimination on the 7900HT Fast Real-Time to a reference gene in a duplex PCR. PCR System. efficient SNP analysis. Between 1 and 3 copies of the gene for Consistent performance—even with the drug-metabolizing enzyme (DME) difficult templates CYP2D6 were detected for 92 human TaqMan® Genotyping Master Mix offers gDNA samples when the samples were unambiguous allelic discrimination amplified using TaqMan® Genotyping even for the most challenging assays. Master Mix (Figure 4). For example, GC-rich targets can pres- 99 ent amplification challenges that reduce SNP detection because of persistent 98 97 TaqMan® Genotyping Master Mix Vendor Si 6.0 6.0 96 5.0 5.0 95 Genotyping ® 4.0 4.0 Call rate (%) 94 ) ) 93 3.0 3.0 AM™ AM™ aqMan Master Mix T e Y (F 92 e Y (F 2.0 2.0 el el All LT B Ep Qi Eu Si All Vendor 1.0 1.0 Figure 2. TaqMan® Genotyping Master 0.0 0.0 Mix provides the highest SNP call rates, outperforming other master mixes. Average -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ® ® call rates for 94 gDNA samples and two NTCs Allele X (VIC ) Allele X (VIC ) genotyped using TaqMan® SNP Genotyping Figure 3. Consistent, reliable SNP detection in a GC-rich region using TaqMan® Genotyping Assay C__27102425_10, with PCR performed Master Mix. Genotyping assays were compared using TaqMan® Genotyping Master Mix and a on the GeneAmp® PCR System 9700 and PCR master mix from vendor “Si” on a set of 94 human gDNA samples (3 ng) and two NTCs, allelic discrimination on the 7900HT Fast using TaqMan® SNP Assay C__12050942_10. PCR was performed on the GeneAmp® PCR Real-Time PCR System. System 9700 and allelic discrimination on the 7900HT Fast Real-Time PCR System. Genotyping Master Mix, combined withTaqMan Mix,combined Genotyping Master copies per diploid genome or one copy per haploid genome. 92 gDNA samples. 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