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Fast, simple SNP genotyping with multiplex qPCR
Thermo Fisher Scientific offers a multiplex SNP genotyping workflow that brings together sample preparation, amplification, and genotype analysis to enable confident multiplexing by real-time PCR. Detect multiple SNP targets in a single reaction to expand assay coverage, generate more data per well, and advance genotyping workflows with coordinated assay and analysis support.
Verified analytical performance for multiplex SNP genotyping
The multiplex, direct-from-lysate SNP genotyping workflow enables analytical performance consistent with purified DNA and singleplex approaches while offering meaningful workflow efficiencies.
Technical Note: Fast, simple SNP genotyping with multiplex qPCR
The analytical evaluation, detailed in the technical note, included crude lysates prepared with the DNA Extract All Reagents Kit and purified genomic DNA tested with TaqMan™ SNP Genotyping Assays, TaqPath ProAmp Master Mix formulations, QuantStudio real-time PCR systems, and Diomni Design and Analysis software.
See how multiplex SNP genotyping enables analytical consistency alongside workflow efficiency:
- 100% concordance between genotype calls from crude lysates and purified genomic DNA for all the SNP targets tested
- 100% concordance between duplex and triplex assay genotyping calls and the same assays run in singleplex format
- Sample preparation reduced to ~5 minutes, with up to 80% fewer hands-on steps compared to column-based purification workflows
Components of the Multiplex SNP Genotyping Workflow
Multiplex SNP genotyping requires five coordinated components verified to support multiplex assay design, direct-from-lysate input, amplification, detection, and data analysis.
Sample preparation reagents
DNA Extract All Reagents Kit generates crude lysates for direct qPCR input, eliminating DNA purification prior to amplification.
- Supports whole blood and buccal swab samples
- Reduces sample preparation time to approximately five minutes
- Minimizes hands-on steps compared to column purification workflows
This direct-from-lysate approach simplifies upstream processing while maintaining compatibility with downstream qPCR.
TaqMan SNP assays for multiplex panel design
TaqMan SNP Genotyping Assays enable laboratories to design and assemble duplex and multiplex SNP panels using our predesigned assay catalog or custom assay designs.
- Select from more than 17 million predesigned human SNP assays
- Design custom TaqMan SNP assays for specific variants or organisms
- Combine selected or custom assays into multiplex SNP panels and assess compatibility with the multiplex interaction check service
This approach allows laboratories to move from target selection to multiplex panel design within a coordinated workflow.
What quencher and dye configurations are available for multiplex TaqMan SNP genotyping assays?
Multiplex SNP genotyping relies on allele-specific probes labeled with distinct reporter dyes to detect multiple targets in a single reaction. Recommended reporter and quencher combinations support 2-SNP and 3-SNP multiplex formats when used with compatible master mixes and instrument configurations. The table below outlines options.
| Assay | Target | Quencher | 5’ reporter dye options |
| 1 | Locus 1 Allele 1 | MGB-NFQ | FAM™ dye VIC™ dye |
| Locus 1 Allele 2 | MGB-NFQ | ||
| 2 | Locus 2 Allele 1 | MGB-NFQ | ABY™ dye Cyanine 5™ (Cy5™) dye |
| Locus 2 Allele 2 | MGB-NFQ | ||
| 3 | Locus 3 Allele 1 | MGB-NFQ | JUN™ dye* |
| Locus 3 Allele 2 | QSY2 | Cyanine 5.5™ (Cy5.5™) dye |
*Requires Custom TaqPath ProAmp Master Mix without ROX dye.
TaqPath ProAmp qPCR Master Mixes
TaqPath ProAmp Master Mix formulations are engineered with an inhibitor-tolerant chemistry designed to support reliable amplification from crude sample lysates in multiplex SNP genotyping workflows.
- TaqPath ProAmp Master Mix (with ROX dye) supports 2-SNP multiplexing (4 targets per reaction)
- Custom TaqPath ProAmp Master Mix without ROX dye supports expanded formats, including 3-SNP multiplexing (6 targets per reaction)
QuantStudio Real-Time PCR Systems
QuantStudio Real-Time PCR Systems are used in this multiplex SNP genotyping workflow to support multiplex dye detection and downstream data analysis.
- Optical channels support multiplex dye combinations, including FAM™, VIC™, ABY™, and Cy5™
- Compatible with duplex and higher-order multiplex assay configurations
- Generate run files required for analysis in Diomni Design and Analysis software
Appropriate instrument configuration is necessary to support multiplex assays.
Diomni Design and Analysis (RUO) Software
Diomni Design and Analysis (RUO) Software supports multiplex SNP genotyping analysis and visualization.
- Automatic genotype calling for duplex and multiplex assay formats
- Multi-plate analysis capability
- Clear allelic discrimination plots for multiplex SNP panels
- Compatible with QuantStudio real-time PCR run files
Manual genotype review for multiplex wells is available in select software builds.
TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. Cy5 is a trademark or registered trademark of GE Healthcare UK Limited.
For Research Use Only. Not for use in diagnostic procedures.