SNP Genotyping is a process by which SNPs can be detected, with several commercially available methods that can be used on a microplate reader, including KASPar®, Taqman®, Amplifluor®, and Invader®. All of these use PCR and primers that are attached to fluorophores. Depending on the changed nucleotide – A, T, G, or C – a different primer with a different fluorophore will be used. Since three distinct fluorophores are used, three different signals have to be measured.
Simultaneous Dual Emission detection significantly improves these SNP assays because two fluorophores can be measured with only one read of the microplate. For instance in the case of KASPar®, the internal standard (ROX) and the allele specific dyes (FAM and VIC) are measured together. Thus, only two measurements have to be taken rather than three, thus reducing the measurement time by 30%. In addition, the misread rate is reduced to <0.5% because plate-plate measurement variation is reduced due to reading the internal standard simultaneously.
To learn more about SNP detection and Simultaneous Dual Emission detection see BMG LABTECH’s application note on KASPar®.
For more information on the PHERAstar Series, visit: http://www.bmglabtech.com/products/microplate-reader/pherastar-series.cfm
|Figure 1. SNP
cluster plot using KASPar® and |
single emission in 1536-well format from KBiosciences'
Genotyping LIMS package.
|Figure 2. SNP cluster plot using KASPar® and |
Simultaneous Dual Emission detection in
1536-well format. Tighter clusters mean less variability.
KASPar is a registered trademark of Kbioscience
Taqman is a registered trademark of Applied Biosystems
Invader is a registered trademark of Third Wave Technologies
Amplifluor is a registered trademark of Millipore