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TaqMan® SNP Genotyping Assays Product Bulletin
Applied Biosystems® TaqMan® SNP Genotyping Assays provide a highly flexible technology for detection of polymorphisms within any genome. TaqMan® Assays have the simplest workflow available and are the quickest way to generate genotyping data. Based on powerful TaqMan® probe and primer chemistry and designs, and coupled to dependable Applied Biosystems® instruments and software, these made-to-order assays produce high-confidence results. TaqMan® Assays are ideal for genotyping applications including screening, association, candidate region, candidate gene, and finemapping studies.
Figure 1. Allelic discrimination is achieved by the selective annealing of TaqMan® MGB probes.
Content-rich marker selection tools simplify study design and help you select from a library of human and mouse assays. This library includes over 4.5 million genome-wide human assays (of which 3.5 million are HapMap SNPbased assays, 160,000 are validated assays, and over 70,000 are coding region assays) and 10,000 mouse assays. We also offer over 2700 inventoried drug metabolism genotyping assays. Additionally, with Custom TaqMan® SNP Genotyping Assays you can confidentially submit target SNP sequences for any genome to create your own assays. Let TaqMan® SNP Genotyping Assays accelerate the pace of your discovery by eliminating time-consuming experimental design and optimization.
Powerful, proven chemistry
Whether your genotyping studies require targeted detection of essential SNPs, or the flexibility to choose SNPs for mapping, TaqMan® SNP Genotyping Assays are the technology of choice. Proven TaqMan® probes, which incorporate minor groove binder (MGB) technology at the 3' end, deliver superior allelic discrimination. The MGB molecule binds to the minor groove of the DNA helix, improving hybridization based assays by stabilizing the MGB probe–template complex.
This increased binding stability permits the use of probes as short as 13 bases for improved mismatch discrimination and greater flexibility when designing assays for difficult or variable sequences. In addition to SNP detection, TaqMan® probes can be designed to detect multiple nucleotide polymorphisms (MNPs) and insertion/ deletions (InDels). Detection is achieved with proven 5' nuclease chemistry by means of exonuclease cleavage of an allele-specific 5' dye label, which generates the permanent assay signal (Figure 1). All MGB probes include a nonfluorescent quencher (NFQ) that virtually eliminates the background fluorescence associated with traditional quenchers and provides a greater signal-to-noise ratio for superior assay sensitivity.
TaqMan® SNP Genotyping Assays collection
TaqMan® SNP Genotyping Assays are the world’s largest collection of single-tube, ready-to-use SNP assays available. The TaqMan® SNP Genotyping Assays library consists of two collections of human assays and one of mouse assays, and can be supplemented with assays designed using our Custom TaqMan® SNP Genotyping Assays Service.
Over 4.5 million human SNP genotyping assays
This assay group contains over 4.5 million genome-wide SNPs, providing unprecedented marker coverage. Included in this collection are 160,000 validated assays that have approximately 10 kb spacing across gene regions. These assays were subjected to an extensive minor allele frequency test in 2–4 ethnic populations (45 individual samples per ethnic group) and, as a result, offer the highest success rate. Also included are over 70,000 assays for the detection of nonsynonymous SNPs in coding regions, including many putative functional SNPs.
Figure 2. Identifying optimal subsets of SNPs with the SNP Wizard. (A) Search and visualize your gene of interest, and then activate the SNP Wizard. (B) Choose a tagging SNP method, and set parameters to reduce the number of SNPs needed to represent common haplotypes. A summary panel appears that displays algorithm results and allows you to easily adjust your parameter selection. Selected tagging SNPs are shown in red on the graphical map.
Over 10,000 mouse SNP genotyping assays
The Mouse TaqMan® Predesigned SNP Genotyping Assays collection consists of over 10,000 assays, and can be supplemented with assays designed using our Custom TaqMan® SNP Genotyping Assays Service.
TaqMan® Drug Metabolism Genotyping Assays
The collection of TaqMan® Drug Metabolism Genotyping Assays includes over 2700 assays that target high-value polymorphisms in more than 221 drug metabolism genes. These assays have proven performance in four different ethnic population samples, consisting of 45 individuals each. To enable easy identification, these assays have been mapped to the common public allele nomenclature websites where possible. Visit dme.appliedbiosystems.com for more information.
All TaqMan® SNP Genotyping Assays are generated using next-generation algorithms from the Applied Biosystems® bioinformatics pipeline. Bioinformatics evaluation of target SNP sequences includes the masking of adjacent SNPs and ambiguous bases so that assay design and subsequent performance is not affected by poor quality of the underlying sequence. Lastly, the assay designs are aligned to the human genome using BLAST to ensure that each assay binds uniquely to the intended polymorphism. As the Custom TaqMan® SNP Genotyping Assay Service is confidential and secure, you simply perform your own bioinformatics analysis prior to submitting your sequence for assay design.
Free marker selection tools:
Custom assay service for any possible SNP
Custom TaqMan® SNP Genotyping Assays can be developed for any SNP in any organism. This service can generate assays for the detection of SNPs, MNPs, or InDels of up to six bases. Custom TaqMan® SNP Genotyping Assays provide you with a complete service that includes secure and confidential ordering, assay design and manufacturing, and quality-control testing for synthesis accuracy and formulation completeness. Additionally, custom human assays are subjected to a functional test on 20 unique DNA samples. Use the free Custom TaqMan® Assay Design Tool to input and submit your sequence for assay design. This easy-to-use online resource lets you quickly submit your sequence information and start the ordering process securely and confidentially. Access the Custom TaqMan® Assay Design Tool at www.appliedbiosystems.com/cadt.
Quality design and manufacturing
Probes and primers used in TaqMan® SNP Genotyping Assays are designed using our rigorous bioinformatics pipeline. This proprietary group of algorithms has generated millions of TaqMan® Assay designs by utilizing heuristic design rules deduced from both manufacturing and assay performance data. All assays are designed to perform under universal reaction conditions, as calculated probe and primer melting temperatures are consistent and include contributions from associated probe conjugates (i.e., dyes and MGB). After manufacturing, assay components undergo extensive laboratory testing at our state-of-the-art manufacturing facility. Quality-control testing includes mass spectrometry for sequence verification and formulation assessments of probe and primer concentrations. Additionally, all human SNP genotyping assays are functionally tested to ensure allelic discrimination.
Simple workflow for quick results
TaqMan® SNP Genotyping Assays constitute the simplest SNP genotyping technology available. We deliver your ready-to-use SNP genotyping assay at ambient temperature in a convenient, single-tube format. The rest is easy. Just combine the assay with TaqMan® Genotyping Master Mix or TaqMan® Universal PCR Master Mix and your purified DNA sample (Figure 3). There is no need to optimize probe, primer, or salt concentrations or temperature because all assays use universal reagent concentrations and thermal cycling conditions.
Figure 3. A simple workflow and reliable instruments combine to generate fast, high-confidence results.
After thermal cycling, no transfers, washes, or additional reagents are required, and the plate remains sealed; just read the plate and analyze the genotypes (Figure 4). This reduces the chance of contamination, sample mix-up, and sample loss. The simplicity of the chemistry allows you to easily automate the reaction for massively parallel genotyping studies, readily increasing the number of assays, number of samples, or both. Additionally, the analysis software allows you to auto-call genotypes, minimizing manual intervention.
Figure 4. TaqMan® Genotyper Software automatically determines sample genotypes and displays data.
Reliable real-time PCR platforms A suite of superior Applied Biosystems® instrument platforms is available for processing and analyzing TaqMan® SNP Genotyping Assays (Table 1). These instruments, which meet all throughput needs and budgets, include the GeneAmp® PCR System 9700 and Veriti® Thermal Cyclers for endpoint PCR, Applied Biosystems® 7500, 7500 Fast, 7900HT Fast, ViiA™ 7, StepOne™, and StepOnePlus™ Real-Time PCR Systems, and the high-throughput OpenArray® Real-Time PCR System. Following PCR amplification, an endpoint read can be performed on any Applied Biosystems® Real-Time PCR System. All of these dependable instruments offer the advanced multicolor detection capabilities required for highly accurate and reproducible allelic discrimination assays.
|Applied Biosystems® GeneAmp® PCR System 9700 Thermal Cycler||60-, 96-, Dual 96-, or Dual 384-well blocks|
|Applied Biosystems® Veriti® Thermal Cycler||60-, 96- (Standard or Fast), or 384-well block|
|Applied Biosystems® 7500/7500 Fast Real-Time PCR System||96-well block (Standard and Fast)|
|Applied Biosystems® 7900HT Fast Real-Time PCR System||384-well block (Standard and Fast)|
|Applied Biosystems® ViiA™ 7 Real-Time PCR System||96- and 384-well blocks (Standard and Fast)|
|Applied Biosystems® StepOne™ Real-Time PCR System||48-well block (Standard and Fast)|
|Applied Biosystems® StepOnePlus™ Real-Time PCR System||96-well block (Standard and Fast)|
|Applied Biosystems® OpenArray® Real-Time PCR System||3072-well plate (Standard)|
Data analysis software
The sophisticated SDS software package provided with all Applied Biosystems® Real-Time PCR Systems facilitates experimental setup, data collection, and assay performance analysis. The SDS software uses an advanced multicomponent algorithm to calculate the distinct signal contribution of each allele of a marker from the fluorescence measurements of each sample well during the assay plate read. The multicomponent data collected from the plate read are stored as SDS files, ready for genotype determination by the SDS software or optional Applied Biosystems® TaqMan® Genotyper Software.
TaqMan® Genotyper Software is a great resource for fast and accurate genotype calling. It is a free SNP genotyping data analysis tool for use with TaqMan® SNP Genotyping Assays performed in 48-, 96-, or 384-well microtiter plates or on OpenArray® Plates. It has a state-of-the-art genotype-calling algorithm, an intuitive user interface, and enhanced study-based analysis features. The software enables multi-plate data analysis for high-throughput workflows and improved accuracy in genotype calling; versatile export features and comprehensive quality-control features facilitate streamlining of the entire workflow. TaqMan® Genotyper Software can be downloaded at www.lifetechnologies.com/taqmangenotyper.
Figure 5. The flexible, high-productivity Applied Biosystems® ViiA™7 Real-Time PCR System.
For large-scale studies, samples can be cycled on multiple Dual 384-Well GeneAmp® 9700 or Veriti® Thermal Cyclers, or Dual Block GeneAmp® PCR 9700 Systems, and analyzed on a ViiA™ 7 System or an OpenArray® Real-Time PCR System, depending on throughput needs. The ViiA™ 7 System (Figure 5) facilitates high-throughput applications by allowing large sample batches (up to eighty-four 384-well plates) to be processed without manual intervention, using the optional Automation Accessory.
Figure 6. The OpenArray® Real-Time PCR System (Figure 6) enables PCR-based SNP genotyping analysis of hundreds to thousands of samples a day across a moderate number of SNPs. Following data collection, you can accelerate and simplify high-volume data transfer and analysis with Applied Biosystems® TaqMan® Genotyper Software, which works with most Applied Biosystems® Real-Time PCR Systems.
Selecting and ordering TaqMan® SNP Genotyping Assays is as simple as “point and click.” Use SNPbrowser™ Software to select the most informative SNPs for your genotyping studies. As you identify SNPs of interest, simply upload your selected TaqMan® SNP Genotyping Assays to the Applied Biosystems website for ordering. The Applied Biosystems website (Figure 7) enables you to search, select, and order from our catalog of over 4.5 million TaqMan® SNP Genotyping Assays. You can search for SNPs using any of several criteria: National Center for Biotechnology Information (NCBI) gene ID, NCBI SNP reference ID (rs#), or gene symbol. You can further refine your search by using SNP type (i.e., intragenic, 5’ or 3’ UTR, chromosome, etc.).
Figure 7. The Life Technologies's website makes ordering easy. For convenient online ordering and multiple search options for all our genotyping assays, including keyword, batch, and location searches, visit the SNP Genotyping Analysis page.
Our Custom TaqMan® SNP Genotyping Assays Service is provided to supply you with SNPs that are not available from our catalog, including those from any nonhuman organism. This service designs assays for all possible SNP, MNP, and InDel targets. Our complementary Custom TaqMan® Assay Design Tool conveniently formats your target sequence for submission to our manufacturing facilities. To order custom assays, simply upload your submission file at www.appliedbiosystems.com/cadt.
TaqMan® Copy Number Assays Product Bulletin
Custom Plus TaqMan® Copy Number Assays, and Custom TaqMan® Copy Number Assays
- Simplest method available to study copy number variation
- Pre-designed human and mouse assays for copy number analysis
——Over 1.6 million pre-designed assays available for genome-wide coverage
——Genes (exons, introns, and junctions)
——Known copy number variations (CNVs)
——Over 180,000 pre-designed assays available
——Gene exon coverage
- Assays available for common vector marker and reporter genes
- Custom Plus TaqMan® Copy Number Assays for user-defined human and mouse genomic targets
——Bioinformatics ensures best possible assay design
——SNPs and repetitive sequence masking
——Genome specificity checks
Custom assays for other targets of interest
——Submit masked target sequences for assay design
——Submit primer/probe pair sequences for assay synthesis
- Reference assays for unique human and mouse genomic sequences
Easy-to-use, robust, and accurate—gold standard TaqMan® Assays designed for the detection and quantitation of copy number variation targets.
Human genomes vary from one another at the genetic level. Some genetic variations are large, structural, chromosomal variations, while others occur at the single-nucleotide level. A structural variation that has been described in recent years is “copy number variation” (CNV). A copy number variation occurs when a DNA segment that is from 1 kb to several megabases in length is present in variable copy numbers compared to a standard, reference genome. There are different types of CNVs, from simple tandem duplications to more complex gains or losses of these sequences at multiple sites throughout the genome (Figure 1). These structural variants are found in all humans as well as other animals and plants.
Figure 1. Types of genomic structural changes affecting segments of DNA that lead to different types of variations.
A fast and simple method
Genome-wide microarray-based technologies are currently available for CNV analysis, but they are not the optimum platform for target-region or validation needs. TaqMan® Copy Number Assays combine TaqMan® Assay chemistry with Applied Biosystems® Real-Time PCR Instruments to form a method for obtaining specific, reproducible, and easy-to-interpret copy number results. This method is fast and simple, and can be completed in hours rather than days. TaqMan® Assays are the gold standard for accurate target quantitation, making them ideal for use in microarray follow-up studies. TaqMan® Copy Number Assays can also be used to screen specific targets, and the workflow can be automated so that several hundred to thousands of samples can be processed in a single day.
The TaqMan® Copy Number Assays family, including TaqMan® Copy Number Assays, Custom Plus TaqMan® Copy Number Assays, and Custom TaqMan® Copy Number Assays, consists of a FAM™ dye–labeled minor groove binder (MGB) probe and unlabeled PCR primers. TaqMan® Copy Number Assays are run simultaneously with a choice of TaqMan® Copy Number Reference Assays (VIC® dye–labeled TAMRA™ probes) in a duplex real-time polymerase chain reaction. The copy number assay detects the target gene or genomic sequence of interest and the reference assay detects a sequence that is known to be present in two copies in the diploid genome. Relative quantitation analysis is performed with CopyCaller™ Software (Figure 2) using either a known calibrator sample or no-calibrator sample method.
The simplest workflow
TaqMan® Copy Number Assays have the simplest workflow of all currently available CNV analysis methods (Figure 3). The test assay (FAM™ dye–labeled), the reference assay (VIC® dye–labeled), your sample DNA, and TaqMan® Master Mix are combined and then run on an Applied Biosystems® Real‑Time PCR System using the standard TaqMan® Genotyping Assay protocol. On average, setup to primary analysis takes only 3–4 hours (including a ~2 hour PCR run).
Figure 2. Determining the copy number with CopyCaller™ Software.
Figure 3. TaqMan® Copy Number Assays have a simple workflow, and on average, setup to primary analysis takes only 3–4 hours (including a ~2 hour PCR run). Each copy number quantitation reaction contains four components: a TaqMan® Copy Number Assay, a TaqMan® Copy Number Reference Assay, TaqMan® Master Mix, and purified genomic DNA sample, run in four replicate wells.
Choose only what you need
With TaqMan® Copy Number Assays, you only choose the assays you need for your project from the large variety of pre-designed assays, or create your own Custom Plus or Custom TaqMan® Copy Number Assays. Because the assay sets are not fixed, follow-up studies can be easily adjusted as your project develops and changes.
TaqMan® Copy Number Assays include pre-designed collections for both human and mouse genomes. The human collection includes more than 1.6 million assays targeting known genes, CNV sequences within the Database of Genomic Variants (DGV) and extragenic/non-gene regions. For most genes, assays are available for each exon of the gene, where possible, plus assays for intron sequences and junctions. The mouse pre-designed collection includes more than 180,000 assays targeting gene exons. Pre-designed assays to common vector marker and reporter genes are also available for transgenic studies.
Custom Plus TaqMan® Copy Number Assays are an optimal solution for studying variation in human and mouse genomic regions of interest for which a pre-designed assay is not available. Custom Plus Assays use the same bioinformatics pipeline used to manufacture pre-designed TaqMan® Copy Number Assays, and can be generated for high-quality genomic targets of interest using the GeneAssist™ Copy Number Assay Tool. A target range is defined by the user on the Genome Map, then, pre-masked targets are created and submitted to our proprietary Copy Number Assay design pipeline. Benefits include genome quality checks and human/mouse reference assay compatibility checks. Users receive Custom Plus Assay annotations that are similar to those for pre-designed assays in the Assay Information Files (AIFs); e.g., gene and DGV locations, genomic location and context sequence. Note that users will not receive their sequence information when they order Custom Plus TaqMan® Copy Number Assays.
Custom TaqMan® Copy Number Assays are an option for additional targets of interest. Custom assays are designed using Applied Biosystems® assay design algorithms, which are optimized to produce high-performing copy number assays. The GeneAssist™ Copy Number Assay Tool enables users to submit their own pre-masked custom target sequences for assay design or primer/probe pair sequences for assay formulation. Custom Assay designs do not go through genome quality checks, but can be compared with the human/mouse reference assays for compatibility in duplex reactions. Users will receive Custom Assay sequences in their AIFs.
TaqMan® Copy Number Reference Assays are available to help perform accurate relative quantitation of copy number target sequences. Two reference assays are available for copy number analysis in humans: TaqMan® Copy Number Reference Assay RNase P (recommended) and TaqMan® Copy Number Reference Assay TERT. This gives users an option in the event that one of the reference assays functions poorly with a sample due to chromosomal aberrations or other issues. Two reference assays are also available for copy number analysis in mice: TaqMan® Copy Number Reference Assay, Mouse, Tfrc (recommended) and TaqMan® Copy Number Reference Assay, Mouse, Tert. Note that the reference assays are species-specific. The reference assays are not primer limited and therefore are highly recommended for copy number analysis.
Powerful data analysis software
CopyCaller™ Software was developed specifically for TaqMan® Copy Number Assay data analysis. This free, easy-to use software utilizes a graphical interface and quickly calculates the possible copy numbers for a set of samples in a run. It also estimates a confidence value for each copy number call and has outlier removal functionality.