Primer Design for Multiplexed Genotyping

Abstract

Single-nucleotide polymorphism (SNP) genotyping can be carried out by annealing an oligonucleotide primer directly adjacent to the polymorphism and carrying out a single base extension using a polymerase reaction with labeled dideoxynucleotide triphosphates. This can be multiplexed by attaching a unique tag at the 5'-end of each oligonucleotide primer and binding the corresponding antitag to a DNA microarray or microbead. After the polymerase reaction, the tag-antitag system can be used to demultiplex the experiment. However, such an assay requires careful primer and tag design to avoid any crossreactivity among the primers, tags, antitags, and template sequence. A procedure for designing the primers is described in this chapter.