Primer-design for multiplexed genotyping.
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.
Kaderali
Lars
L
ZAIK, University of Cologne, Weyertal 80, 50931 Cologne, Germany. kaderali@zpr.uni-koeln.de
Deshpande
Alina
A
Nolan
John P
JP
White
P Scott
PS
D000465:Algorithms; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012984:Software
D017931:DNA Primers; D009841:Oligonucleotides