A high-throughput SNP typing system for genome-wide association studies

Citation
Y. Ohnishi et al., A high-throughput SNP typing system for genome-wide association studies, J HUM GENET, 46(8), 2001, pp. 471-477
Citations number
10
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Molecular Biology & Genetics
Journal title
JOURNAL OF HUMAN GENETICS
ISSN journal
1434-5161 → ACNP
Volume
46
Issue
8
Year of publication
2001
Pages
471 - 477
Database
ISI
SICI code
1434-5161(2001)46:8<471:AHSTSF>2.0.ZU;2-T
Abstract
One of the most difficult issues to be solved in genome-wide association st udies is to reduce the amount of genomic DNA required for genotyping. Curre ntly available technologies require too large a quantity of genomic DNA to genotype with hundreds or thousands of single-nucleotide polymorphisms (SNP s). To overcome this problem, we combined the Invader assay with multiplex polymerase chain reaction (PCR), carried out in the presence of antibody to Taq polymerase, as well as using a novel 384-well card system that can red uce the required reaction volume. We amplified 100 genomic DNA fragments, e ach containing one SNP, in a single tube, and analyzed each SNP with the In vader assay. This procedure correctly genotyped 98 of the 100 SNP loci exam ined in PCR-amplified samples from ten individuals; the genotypes were conf irmed by direct sequencing. The reproducibility and universality of the met hod were confirmed with two additional sets of 100 SNPs. Because we used 40 ng of genomic DNA as a template for multiplex PCR, the amount needed to ass ay one SNP was only 0.4ng; therefore, theoretically, more than 200,000 SNPs could be genotyped at once when 100 mug of genomic DNA is available. Our r esults indicate the feasibility of undertaking genome-wide association stud ies using blood samples of only 5-10ml.