Primer‐design for multiplexed genotyping

• Published in: Nucleic acids research 2003-03, Vol.31 (6), p.1796-1802
• Main Authors: Kaderali, Lars, Deshpande, Alina, Nolan, John P., White, P. Scott
• Format: Article
• Language: English
• Subjects: Algorithms; Base Sequence; DNA Primers - genetics; Genome, Human; Genotype; Humans; Molecular Sequence Data; Oligonucleotides - genetics; Polymorphism, Single Nucleotide - genetics; Software
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkg267

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 flow 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 high‐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.