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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
11
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pubmed:dateCreated |
2001-11-5
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pubmed:abstractText |
The emerging role of single-nucleotide polymorphisms (SNPs) in clinical association and pharmacogenetic studies has created a need for high-throughput genotyping technologies. We describe a novel method for multiplexed genotyping of SNPs that employs PCR amplification on microspheres. Oligonucleotide PCR primers were designed for each polymorphic locus such that one of the primers contained a recognition site for BbvI (a type IIS restriction enzyme), followed by 11 nucleotides of locus-specific sequence, which reside immediately upstream of the polymorphic site. Following amplification, this configuration allows for any SNP to be exposed by BbvI digestion and interrogated via primer extension, four-color minisequencing. Primers containing 5' acrylamide groups were attached covalently to the solid support through copolymerization into acrylamide beads. Highly multiplexed solid-phase amplification using human genomic DNA was demonstrated with 57 beads in a single reaction. Multiplexed amplification and minisequencing reactions using bead sets representing eight polymorphic loci were carried out with genomic DNA from eight individuals. Sixty-three of 64 genotypes were accurately determined by this method when compared to genotypes determined by restriction-enzyme digestion of PCR products. This method provides an accurate, robust approach toward multiplexed genotyping that may facilitate the use of SNPs in such diverse applications as pharmacogenetics and genome-wide association studies for complex genetic diseases.
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/11691857-10084106,
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http://linkedlifedata.com/resource/pubmed/commentcorrection/11691857-9862993
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Nov
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pubmed:issn |
1088-9051
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
11
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
1926-34
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
pubmed-meshheading:11691857-DNA,
pubmed-meshheading:11691857-Fluorescent Dyes,
pubmed-meshheading:11691857-Gene Amplification,
pubmed-meshheading:11691857-Genome, Human,
pubmed-meshheading:11691857-Genotype,
pubmed-meshheading:11691857-Humans,
pubmed-meshheading:11691857-Microspheres,
pubmed-meshheading:11691857-Nucleic Acid Amplification Techniques,
pubmed-meshheading:11691857-Nucleic Acid Heteroduplexes,
pubmed-meshheading:11691857-Polymerase Chain Reaction,
pubmed-meshheading:11691857-Polymorphism, Single Nucleotide,
pubmed-meshheading:11691857-Sequence Analysis, DNA
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pubmed:year |
2001
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pubmed:articleTitle |
SNP genotyping by multiplexed solid-phase amplification and fluorescent minisequencing.
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pubmed:affiliation |
Affymax Inc., Palo Alto, California 94304, USA.
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pubmed:publicationType |
Journal Article
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