Staged oligonucleotide design, compilation and quality control procedures for multiple SNP genotyping by Multiplex PCR and Single Base Extension Microarray format

Abstract

The value of the SNPs (Single Nucleotide Polymorphisms) as markers in various studies depends on their absolute numbers, but also on the more complicated issue of their availability. For any given study there must be enough SNPs suitably placed and satisfying specific generic qualitative criteria. The feasibility of a study depends on the ability to replace and keep the number of marker loci reasonably stable at each phase of the selected experimental procedures during the development of the overall methodology. We embarked on an experimental simulation, where we specified a series of criteria for the number and qualities of SNPs needed for a hypothetical study and proceeded to SNP selection and computerized primer design for each SNP locus. The decisive factor was the applicability of the primers in multiplex PCR format. The editing steps and software upgrades of the adopted selection procedure resulted in 98.6% primer efficiency. A total of 148 primer pairs were designed fulfilling multiplex PCR specifications, which were also effective in simplex quality control PCR assays against a start-up number of 150