Abstract
The need for large-scale and high-throughput methods for SNP genotyping has rapidly increased during the last decade. Our system, presented here, combines the highly specific genotyping principle of minisequencing with the advantages of a microarray format that allows highly multiplexed and parallel analysis.
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 5′ tag sequences 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; then the fluorescent signals from the array are measured, and the genotypes are deduced according to the label incorporated. The “array of arrays” format of the system, accomplished by a silicon rubber grid giving separate reaction chambers, allows either 80 or 14 samples to be analyzed for up to 200 or 600 SNPs, respectively, on a single microscope slide.
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Acknowledgment
This protocol is the result of the combined work effort of both former and present members of the Molecular Medicine research group at the Department of Medical Sciences at Uppsala University. The group has received financial support from the Swedish Research Council, the Wallenberg Foundation, and the European Commission (FP5 and FP6).
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Lovmar, L., Syvänen, AC. (2005). Genotyping Single-Nucleotide Polymorphisms by Minisequencing Using Tag Arrays. In: Joos, T.O., Fortina, P. (eds) Microarrays in Clinical Diagnostics. Methods in Molecular Medicine™, vol 114. Humana Press. https://doi.org/10.1385/1-59259-923-0:79
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DOI: https://doi.org/10.1385/1-59259-923-0:79
Publisher Name: Humana Press
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Online ISBN: 978-1-59259-923-3
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