Kaderali, Lars;Deshpande, Alina;White, P. Scott - SBEprimer: Primer Design for Multiplexed Genotyping

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Title: SBEprimer: Primer Design for Multiplexed Genotyping	P73
Kaderali, Lars; Deshpande, Alina; White, P. Scott kaderali@zpr.uni-koeln.de ZAIK, University of Cologne, Germany/Los Alamos National Laboratories, USA

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) allows easy genotyping of single mutation sites [3]. The idea is to anneal an oligonucleotide primer directly adjacent to the polymorphic site, and use a polymerase reaction to append a single labelled ddNTP base directly opposite the mutation. The label identifies the base inserted, and thus the mutation present at the interrogated locus.

The technology can be massively parallelised using DNA microchips or flow cytometry with microspheres as solid support [1, 2]. By adding a unique oligonucleotide tag to the 5' end of the minisequencing primer and attaching the complementary anti-tag to the chip or bead surface, the assay can be "demultiplexed": Each site-specific primer is "sorted" to a specific spot on the chip or to a specific color-coded bead, the position on the chip (the color of the bead, respectively) identifying the polymorphic site, and the flourescent label of the ddNTP inserted in the polymerase reaction showing the mutation present.

Such high-throughput SNP scoring requires a high level of primer multiplexing in order to analyse multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring.

Primers can be chosen from either the plus or the minus strand, and primers used in the same experiment must not bind to one another. To genotype a given number of polymorphic sites, the question is which primer to use for which SNP, and which primers to group together into the same experiment. Furthermore, a crosshybridisation-free tag/anti-tag code is required in order to sort the extended primers to the corresponding microspheres or chip spots. These problems pose challenging algorithmic questions.

We use a dynamic programming algorithm to calculate the free energy of DNA-DNA interactions. This algorithm is used to evaluate primer candidates and check for false priming, hairpin and primer dimer formation, as well as in the generation of a tag/anti-tag code. The problem of choosing appropriate sets of crosshybridisation free polymerase primers to work together in one experiment is modelled as a generalized graph coloring problem.

We present a computer program to automate the design process for the assay and solve the above mentioned problems. 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 tags is automatically done in a way to avoid 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 if assay design is carefully undertaken.

[1] Cai, H.; White, P.S.; Torney, D.C.; Deshpande, A.; Wang, Z.; Marrone, B.; Nolan, J. Flow cytometry-based minisequencing: A new platform for high-throughput single-nucleotide polymorphism scoring. Genomics 66, 135-143.
[2]: Hirschhorn, J.N.; Sklar, P.; Lindblad-Toh, K.; Lim, Y.-M.; Ruiz-Gutierret, M.; Bolk, S.; Langhorst, B.; Schaffner, S.; Winchester, E.; Lander, E.S. Sbe-tags: An array-based method for efficient single-nucleotide polymorphism genotyping. PNAS 97, 12164-12169.
[3]: Syvännen, A.-C. From gels to chips: "minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms. Human Mutation 13, 1-10.