Detection of human apolipoprotein E3, E2, and E4 genotypes by an allele-specific oligonucleotide-primed polymerase chain reaction assay: development and validation.
A polymerase chain reaction (PCR) assay has been developed and validated by using allele-specific oligonucleotide (ASO) primers to specifically amplify E3, E2, and E4 polymorphic sequences of the human apolipoprotein E (apo E) genes. Degenerate ASOs containing one or two additional 3' mismatches provided greater specificity than did ASOs containing a single mid-sequence or 3' allele-specific mismatch with plasmid pEB4 or genomic DNA as template. Optimal specificity and efficiency of amplification did not correlate with primer annealing conditions, whether determined theoretically or via oligo-melting experiments. Pre-cycling denaturation times and high cycling denaturation temperatures were also required for optimal amplification, presumably because of the high G:C content (75-85%) of apo E gene sequences. Conditions permissive for amplification and discrimination with plasmid DNA did not transpose favorably to amplification from human genomic DNA from peripheral blood leukocytes; the latter required nested primer reactions. These data may be valuable in predicting PCR assay conditions for other G:C-rich sequences containing polymorphic sequence differences. The assay described is both more accurate and rapid (24 h) than previously described methods for phenotyping or genotyping human apo E from blood specimens.
Green
E K
EK
University Department of Clinical Chemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, U.K.
Bain
S C
SC
Day
P J
PJ
Barnett
A H
AH
Charleson
F
F
Jones
A F
AF
Walker
M R
MR
D000483:Alleles; D053329:Apolipoprotein E2; D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013698:Templates, Genetic
D053329:Apolipoprotein E2; D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E; D009841:Oligonucleotides; D004247:DNA