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Nucleic Acids Research, 2003, Vol. 31, No. 6 1796-1802
© 2003 Oxford University Press

Primer-design for multiplexed genotyping

Lars Kaderali, Alina Deshpande1, John P. Nolan1 and P. Scott White1

ZAIK, University of Cologne, Weyertal 80, 50931 Cologne, Germany and 1 Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA

*To whom correspondence should be addressed. Tel: +49 221 470 6003; Fax: +49 221 470 5160; Email: kaderali{at}zpr.uni-koeln.de

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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