What does duplexing a genotyping assay mean?

In an Applied Biosystems TaqMan SNP genotyping assay there is a forward and reverse primer and two probes that are labelled with different fluorescent dyes, one for each allele.  Duplexing a genotyping assay means there will be two SNP genotyping assays in a single well. When you multiplex a genotyping assay, you test for the presence of two SNP alleles at two different locations in a genome, using the same samples and reagents in the same tube or well at the same time.
While duplexing is more commonly done with gene expression assays, duplexing can also be done with genotyping assays.

Duplexing or multiplexing real-time PCR assays for genotyping increases efficiency by saving samples, reagents, and time.


Dyes, reagents, and software

In a singleplex genotyping assay, each well must contain two probes labeled with different fluorescent dyes—one to detect each variant. Therefore, in a duplex genotyping reaction, each well will consist of four fluorescent dyes to amplify allele 1 of SNP A, allele 2 of SNP A, allele 1 of SNP B, and allele 2 of SNP B.

If we are to clearly distinguish the signals from the four dyes, their spectra must peak at distinctly different points on the visible spectrum. Thermo Fisher Scientific offers two reporter dyes, ABY (yellow) and JUN (orange-red), that complement the widely used FAM (blue) and VIC (green) dyes. This set of four dyes, with minimaly overlapping emission spectra, is ideal for duplex genotyping.

Figure 1.
Figure 1. Fluorescence emission spectra of different dyes used for multiplex qPCR.

Researchers using this technology should be aware that each of the millions of pre-designed human and mouse genotyping assays that we offer uses the FAM and VIC dyes with an MGB-NFQ quencher. Assays using the ABY and JUN dyes need to be designed with a longer probe to accommodate a different quencher, QSY, to avoid possible inhibition of PCR by MGB.

You will also need to use a Master Mix of enzymes, nucleotides, and other reagents that is compatible with all four dyes. Applied Biosystems TaqMan Multiplex Master Mix and TaqPath ProAmp Multiplex Master Mix are formulated for multiplexing success. They contain Mustang Purple dye, a passive reference dye detected by the fifth filter that is used for normalization in place of the ROX dye detected by the fourth filter. As the name implies, the fluorescence of this dye peaks in the far-red region of the visible spectrum, and has minimal overlap with the four dye choices mentioned above that are bound to the probes. These master mixes for multiplexing contain higher enzyme and dNTP concentrations and produce accurate and precise results in duplex genotyping assays. 

For data analysis, most of the programs available for genotyping applications using real-time PCR assume that there are only two dyes in each tube or well, and that these are FAM and VIC. If you use an Applied Biosystems real-time PCR system, you have a readily available solution that can handle the four fluorophores on the Thermo Fisher Cloud (www.thermofisher.com/cloud). Scroll down to ‘Data Analysis’ and then click ‘qPCR’ to learn more about the genotyping (GT) app and other available software options.



Below is an example of data generated from duplexing two custom TaqMan SNP genotyping assays targeting biallelic SNPs in maize.  The assays were designed using the Custom TaqMan Assay Design Tool (thermofisher.com/snpcadt).

Crude lysates from maize seed chips were generated with the DNA Extract All Reagents Kit and used directly in qPCR by adding TaqMan Multiplex Master Mix.  A typical genotyping experiment was performed using two assays in each well, and the standard genotyping experimental method was used on the QuantStudio 5 Real-Time PCR System.

The trajectory of DNA samples by cycle (gray lines) allows determination of the number of cycles that gives the best combination of allelic discrimination and signal intensity. This feature also helps identify genotypes of undetermined or ambiguous samples by showing the trajectory of the curve from a sample relative to those of other samples within genotype clusters. The genotype calls shown were made automatically by the GT App on the Thermo Fisher cloud.

Figure 2 shows two allelic discrimination plots from the Thermo Fisher Cloud GT interface. The data from both plots were generated from assays that were run in the same well.  Gray lines showing the path of each sample through cycle 50 were generated using real-time PCR data.

Figure 2a
Figure 2A. Assay 1. Allele 1 was detected by the probe labeled with VIC dye and the Allele 2 probe was labeled with FAM dye.
Figure 2a
Figure 2B. Assay 2. Allele 1 was detected by the JUN-labeled probe and Allele 2 was detected by the ABY-labeled probe.