Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers.
A method for apolipoprotein (apo) E genotyping was developed using the polymerase chain reaction (PCR) with allele-specific oligonucleotide primers (ASP). Synthetic oligonucleotides with base-pair mismatches at the 3' terminus were used as primers to amplify the apoE gene in subjects previously phenotyped using isoelectric focusing (IEF). Complementary primer-allele combinations were specifically amplified by PCR, together with a control pair of primers specific to the human prothrombin gene. Identification of genotype by PCR using ASP was consistent with the phenotypes that were determined by IEF for 14 healthy normolipidemic subjects. These results were achieved using DNA isolated from buccal epithelial cells obtained from a mouthwash or DNA extracted from leukocytes. Genotype identification required analysis of the PCR products on an ethidium-stained agarose gel, yielding results 3 h after DNA extraction. In comparison with other current methods, PCR using ASP is suggested as a rapid and simple noninvasive technique for determining population apoE allelic distribution.
Main
B F
BF
Division of Human Nutrition, University of British Columbia, Vancouver, Canada.
Jones
P J
PJ
MacGillivray
R T
RT
Banfield
D K
DK
D000483:Alleles; D000595:Amino Acid Sequence; D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic
D001057:Apolipoproteins E; D009841:Oligonucleotides; D004247:DNA