Primer-design for multiplexed genotyping

L. Kaderali
2003 Nucleic Acids Research  
Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Mutation analysis by polymerase-mediated single-base primer extension (minisequencing) can be massively parallelized using DNA microchips or¯ow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5¢ end of the minisequencing primer and attaching the complementary antitag to the array or bead surface, the assay can be'demultiplexed'. Such
more » ... h-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. We present a computer program to automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/antitag system is generated, and the pairing of primers and DNA tags is automatically done in a way to avoid any crossreactivity. We report results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping. The software is available to academic users on request.
doi:10.1093/nar/gkg267 pmid:12626722 pmcid:PMC152869 fatcat:ounwe47xsjaa7n4tnx6d3wkr7a