Primer-Design for multiplexed, minisequencing-based 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 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 have developed 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 results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping.

Publication:")?> This work has been published in Nucleic Acids Research
Kaderali,L., Deshpande,A., Nolan,J.P. and White,P.S. (2003): Primer-Design for Multiplexed Genotyping, Nucleic Acids Research 31 (6), 1796-1802 Software:")?> The software is available for free for academic use, please cite the Nucleic Acids Research Publication. Commercial entities, please contact Lars Kaderali before downloading and using the code. The following programs are available for Unix systems:

• sbeprimer - program to choose minisequencing primers
• taggen - software to generate tag-/antitag-pairs
• align - simply tool that will generate a multiple sequence alignment with no gaps
• dGalign - will calculate an alignment of two sequences, minimizing free energy of the alignment at a giben temperature
• thermAlign - will calculate an alignment of two sequences, maximizing melting temperature of the aligned duplex, given the total molar concentration of the strands and a correction factor for salt conditions
• allpairg - works similar to dgAlign, but compares not just interactions of two strands, but, given a list of DNA sequences, it will calculate all pairwise interactions and write them to a file
• allpairt - works similar to thermalign, but considers all pairwise interactions for a whole set of sequences

Supplementary data for Nucleic Acids Research Publication: