An Assay for Variable Fragment Length Allele-Specific Genotyping

Polymerase chain reaction (PCR) is a quick and easy technique used to detect nucleotide polymorphisms and sequence variations in various fields such as medicine, agriculture, and basic research. There exists a set of PCR variants, collectively called allele-specific PCR, which use competitive reactions in the presence of allele-specific primers to amplify only specific alleles. In this chapter, we present protocols for a bioinformatics tool that can be used to design PCR-based genotyping assays for variable fragment length allele-specific genotyping. This assay is designed in both directions and can target single-nucleotide polymorphisms and insertion/deletions. This chapter provides a step-by-step guide to design a multiplexed primer assay for variable fragment length allele-specific genotyping.