High Speed FRET based SNP Genotyping Measurement on the PHERAstar

Single nucleotide polymorphisms (SNPs) are a marker system that can differentiate individuals based on variations detected at the level of a single nucleotide base in the genome. Such variations are present in large abundance in the genomes of higher organisms such as plants and animals. Here we employ the PHERAstar plate reader and the KASPar^TM system from KBiosciences to measure both SNP genotyping chemistry and high throughput production genotyping. The KASPar^TM approach employs a novel form of allele specific PCR with a format for homogeneous fluorescence resonance energy transfer. Following optimization of gain, excitation and emission filter choice, and number of flashes per well we compared the approach using single and dual emission detection. The results exhibited sharper clusters using two photo-multiplier tubes, indicative of improved accuracy, and a mis-read rate that has decreased from 2-3% to <0.1%. An additional benefit is a 30% reduction in read times. We conclude that pairing the KASPar^TM approach with PHERAstar instrumentation results in increased robustness, discriminating power, accuracy and speed, which will allow this approach to be employed in high throughput screening. The PHERAstar can also be used to measure and increase throughput of other SNP Genotyping chemistries like Taqman® and Invader.