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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
4
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pubmed:dateCreated |
1994-10-10
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pubmed:abstractText |
Genotype determination using the solid-phase minisequencing method of Syvänen et al. (1990, 1993) has been adapted for use with fluorescein-labeled dideoxynucleotides (F-ddNTPs). PCR is performed using one biotinylated primer and one unbiotinylated primer. The biotinylated products are captured in streptavidin-coated microtiter wells. Following removal of nonbiotinylated strands with NaOH, the bound strands are hybridized with a primer adjacent to the polymorphic site being tested. Using T7 DNA polymerase, the primer is extended using one F-ddNTP in the presence of the other three unlabeled ddNTPs. Incorporation of the F-ddNTP is detected by binding antifluorescein antibody conjugated with alkaline phosphatase followed by incubation with a chromogenic substrate. This assay was used to determine APOE genotypes for 75 subjects. The APOE genotypes were also determined using a method involving the incorporation of mobility-shifting nucleotide analogs (Livak et al., 1992). Investigation of the one discrepancy between the two methods revealed that one subject carries a rare APOE allele that has a 3 bp deletion.
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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:issn |
1059-7794
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
3
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pubmed:geneSymbol |
APOE
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
379-85
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pubmed:dateRevised |
2004-11-17
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pubmed:meshHeading |
pubmed-meshheading:8081392-Alleles,
pubmed-meshheading:8081392-Apolipoproteins E,
pubmed-meshheading:8081392-Bacterial Proteins,
pubmed-meshheading:8081392-Base Sequence,
pubmed-meshheading:8081392-Biotin,
pubmed-meshheading:8081392-DNA Primers,
pubmed-meshheading:8081392-Deoxyribonucleotides,
pubmed-meshheading:8081392-Enzyme-Linked Immunosorbent Assay,
pubmed-meshheading:8081392-Fluoresceins,
pubmed-meshheading:8081392-Fluoroimmunoassay,
pubmed-meshheading:8081392-Genotype,
pubmed-meshheading:8081392-Humans,
pubmed-meshheading:8081392-Male,
pubmed-meshheading:8081392-Molecular Sequence Data,
pubmed-meshheading:8081392-Polymerase Chain Reaction,
pubmed-meshheading:8081392-Polymorphism, Genetic,
pubmed-meshheading:8081392-Sequence Analysis, DNA,
pubmed-meshheading:8081392-Streptavidin,
pubmed-meshheading:8081392-Titrimetry
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pubmed:year |
1994
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pubmed:articleTitle |
A microtiter plate assay for determining apolipoprotein E genotype and discovery of a rare allele.
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pubmed:affiliation |
Research & Development Division, Du Pont Merck Pharmaceutical Company, Wilmington, Delaware 19801.
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pubmed:publicationType |
Journal Article
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