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57 Life Technologies ™ | Genome editing Figure 16. TaqMan ® SNP Genotyping Assay. (A) TaqMan ® SNP Genotyping Assays workflow. Genotyping is performed on the QuantStudio ® 12K Flex Real-Time PCR System using the TaqMan ® SNP Genotyping Assay and the 400-bp amplicon containing the region targeted for TAL editing. Results are analyzed with the TaqMan ® Genotyper ™ Software. (B) TaqMan ® SNP Genotyping Assays technology. Discrimination between two SNP alleles (or allelic discrimination) is achieved by selective annealing of two TaqMan ® probes, with each probe specific for one allele and carrying a different fluorescent reporter. During PCR amplification, the probe hybridized on its target allele degrades and generates a specific fluorescence signal. The signal from each reporter is recorded during real-time PCR and plotted on an allelic discrimination plot: the relative fluorescence signal from allele 1 is plotted along the x-axis of the allelic discrimination plot and the relative fluorescence signal from allele 2 is plotted along the y-axis. Colonies homozygous for allele 1 will plot along the x-axis. Colonies homozygous for allele 2 will plot along the y-axis. Heterozygous colonies will plot on a diagonal. A B Design/Order TaqMan ® Genotyping Assay Load master mix, assays, and samples into 96-well plate Perform real-time PCR Analysis using TaqMan ® Genotyper ™ Software V F F V V F F V Homozygous allele 2 Heterozygous No template control Homozygous allele 1 Allelic discrimination plot Relative fluorescent signal from allele 1 Relative fluorescent signal from allele 2 Allelic discrimination achieved by selective annealing of TaqMan ® probes PrOducT BullETIN TaqMan ® Genotyping Assays Introduction Genomic sequencing of a large number of organisms has produced a tremendous amount of information enabling further advances in genomic analysis, including SNP genotyping. Applied Biosystems Custom TaqMan ® SNP Genotyping Assays is a design service that provides optimized, ready- to-run assays for any genome, any organism. These custom assays are designed, synthesized, formulated, quality control tested, and delivered in a convenient, single-tube format. Custom assays use TaqMan ® minor groove- binding (MGB) probe-based assays for superior allelic discrimination and assay design flexibility. Simply submit your target DNA sequence information from any organism, and we will develop your ready-to-use SNP genotyping assay. Custom assays eliminate the time- consuming manual steps of probe and primer design, formulation, and testing to simplify your genomic project plan. The easy workflow entails adding an assay plus TaqMan ® Universal PCR Master Mix or TaqMan ® Genotyping Master Mix with enzyme to your DNA sample and one-step PCR amplification. TaqMan ® Assays enable you to focus on your strain identification, disease association, marker selection, quanti- tative trait loci, linkage mapping, and other SNP genotyping studies. Assay design Service The Custom TaqMan SNP Genotyping Assays Service provides secure and confidential access to Applied Biosystems' assay design expertise. The service uses the proven Applied Biosystems Assay Design Pipeline to create assays designed to your specific- ations. This versatile resource provides genotyping assays for any possible SNP, MNP (up to six base pairs in length), and InDel (up to six base pairs in length). Applied Biosystems synthesizes and tests all assay components in our state- Custom TaqMan ® SNP Genotyping Assays Simplify Your Genomic Projects • Any genome, any organism • Versatility to detect single nucleotide polymorphisms (SNPs), insertion/ deletions (InDels), and multi-nucleotide polymorphisms (MNPs) • Proven TaqMan ® MGB probe-based assays • Made to your specifications • One tube, one seamless workflow