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Clinical Chemistry 37: 1263-1268, 1991;
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Clinical Chemistry, Vol 37, 1263-1268, Copyright © 1991 by American Association for Clinical Chemistry

Detection of human apolipoprotein E3, E2, and E4 genotypes by an allele- specific oligonucleotide-primed polymerase chain reaction assay: development and validation

EK Green, SC Bain, PJ Day, AH Barnett, F Charleson, AF Jones and MR Walker
University Department of Clinical Chemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, U.K.

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.


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