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

Green, Elaine Karen, Bain, S.C., Barnett, A.H., Charleson, F., Jones, A.F. and Walker, M.R. 1991. Detection of human apolipoprotein E3, E2, and E4 genotypes by an allele-specific oligonucleotide-primed polymerase chain reaction assay: development and validation. Clinical Chemistry 37 (7) , pp. 1263-1268.

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Abstract

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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	MRC Centre for Neuropsychiatric Genetics and Genomics (CNGG) Medicine
Subjects:	R Medicine > R Medicine (General)
Publisher:	American Association for Clinical Chemistry
ISSN:	0009-9147
Last Modified:	03 Dec 2015 13:26
URI:	https://orca.cardiff.ac.uk/id/eprint/81113

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