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High-throughput SNP mapping by tag-array minisequencing

A promising approach towards high-throughput genotyping of SNPs is to use arrays of immobilised oligonucleotides in miniaturised assays. Significant advantages of performing the assays in microarray formats are the reduced costs of genotyping due to the simultaneous analysis of many SNPs in each sample, and the small reaction volumes employed. To facilitate positional cloning in Drosophila, we established a microarray system for a genome-wide genotyping panel of 300 SNPs, discriminating between each of the major chromosomes from commonly used laboratory strains of Drosophila. The Information for SNP selection and assay design was taken from the high-density SNP map. After assay development, sets of 60 markers from each chromosomal arm were selected, which resulted in genotyping assays for SNPs at an average spacing of less than 400kb.

The genotyping system is based on multiplex, four-color fluorescent minisequencing. The minisequencing reaction uses a DNA polymerase to extend detection primers that anneal immediately adjacent to the sites of the SNPs. The primers are extended with fluorescently labeled, terminating nucleotide analogues that are complementary to the nucleotide at the SNP site. The reactions are performed in solution using detection primers with 5 -Tag sequences. Each SNP has its own specific tag that is complementary to one of the cTags that are immobilised to the microarray.

When the extended detection primers are applied to the microarray, the tags will hybridise to their corresponding cTags. From the known locations of the cTags on the microarray, the genotypes of the SNPs can be deduced. By the array of arrays format of our method separate reaction chambers are formed on an array by a silicon rubber grid. In this manner a single standard microscope slide can be used to analyse up to 200 SNP positions in 80 samples simultaneously. The tag-array minisequencing method is particularly suitable for large-scale gene mapping in Drosophila due to its robustness, simplicity in assay design and low cost. To facilitate data analysis, IMP/IMBA has developed software, which automatically translates raw data from the microarray analysis to the genotypes. In addition, it provides a user-friendly, graphical output, which simplifies localization of the region of interest.

The tag-array minisequencing system is based on standard instruments, which makes the system easily implemented at other laboratories. MEDSCI has an extensive experience from using the technique, particularly in human genetic applications. MEDSCI also has experiences from organizing practical courses on the technique. In the context of the FLYSNP project MEDSCI organized the First FLYSNP workshop in November 2004. Currently, MEDSCI provides three annual courses for graduate students which include the tag-array minisequencing system.

These courses, one of which is sponsored by EMBO (the European Molecular Biology Organization), are open to interested Drosophila researchers. Moreover, MEDSCI can disseminate the technology by accepting visitors for training in our lab in Uppsala. Genotyping services for the Drosophila community can be arranged on collaborative basis on a small scale. If the interest in gene mapping is large enough, a genotyping service can be established at the existing national SNP genotyping service unit, which operates in close connection with the Molecular Medicine research group at MEDSCI. For more information, please visit

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Ann-christine SYVÄNEN, (Professor)
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