Kaderali, Lars and Deshpande, Aline and Nolan, John P. and White, P. Scott
Primer Design for Multiplexed Genotyping.
Nucleic Acids Research Vol. 31 (6).
Single-nucleotide polymorphism (SNP) ana-lysis 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 anti-tag 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/anti-tag system is generated, and the pairing of primers and DNA-Tags is automatically done in a way to avoid any crossreactivity. We report first results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping.
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