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Primer-design for multiplexed genotyping.

Lars Kaderali·Alina Deshpande·John P Nolan·P Scott White
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Abstract

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.

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AlgorithmsBase SequenceDNA PrimersGenome, HumanGenotypeHumansMolecular Sequence DataOligonucleotidesPolymorphism, Single NucleotideSoftwareJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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BibTeXRIS

Lars Kaderali, Alina Deshpande, John P Nolan, P Scott White. 2003-03-15. Primer-design for multiplexed genotyping.. https://doi.org/10.1093/nar%2Fgkg267

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