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Genotyping Single Nucleotide Polymorphisms by Multiplex Minisequencing Using Tag-Arrays

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DNA Microarrays for Biomedical Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 529))

Abstract

The need for multiplexed methods for SNP genotyping has rapidly increased during the last decade. We present here a flexible system that combines highly specific genotyping by minisequencing single-base extension with the advantages of a microarray format that allows highly multiplexed and parallel analysis of any custom selected SNPs.

Cyclic minisequencing reactions with fluorescently labeled dideoxynucleotides (ddNTPs) are performed in solution using multiplex PCR product as template and detection primers, designed to anneal immediately adjacent and upstream of the SNP site. The detection primers carry unique Tag-sequences at their 5′ ends and oligonucleotides complementary to the Tag-sequence, cTags, are immobilized on a microarray. After extension, the tagged detection primers are allowed to hybridize to the cTags, and the fluorescent signals from the array are measured and the genotypes are deduced by cluster analysis of the incorporated labels. The “array of arrays” format of the system, accomplished by a silicon rubber grid to form separate reaction chambers, allows either 80 or 16 samples to be analyzed for up to 200 or 600 SNPs, respectively on a single microscope slide.

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Acknowledgments

This protolcol is the result of the combined work effort of both former and present members of the research group in Molecular Medicine at the Department of Medical Sciences at Uppsala University. The group has received financial support from the Swedish Research Council, the Wallenberg Foundation, the Swedish Cancer Foundation, and the European Commission.

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  1. Uppsala University, Uppsala, Sweden

    Lili Milani

  2. University Hospital, Uppsala, Sweden

    Ann-Christine Syvänen

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  1. Lili Milani
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  2. Ann-Christine Syvänen
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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Milani, L., Syvänen, AC. (2009). Genotyping Single Nucleotide Polymorphisms by Multiplex Minisequencing Using Tag-Arrays. In: Dufva, M. (eds) DNA Microarrays for Biomedical Research. Methods in Molecular Biology, vol 529. Humana Press. https://doi.org/10.1007/978-1-59745-538-1_14

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    • DOI: https://doi.org/10.1007/978-1-59745-538-1_14

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    • Publisher Name: Humana Press

    • Print ISBN: 978-1-934115-69-5

    • Online ISBN: 978-1-59745-538-1

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