Increase speed and decrease misread rate of SNP detection assays
Single Nucleotide Polymorphisms or SNPs are changes that occur in genomic DNA at one nucleotide that make it different from other members of its species or paired chromosomes. A SNP can change the functionality of a protein, if it falls within a coding region, or it can be used as a genetic marker to identify a distinct subspecies population.
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.
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