High sensitivity of laser-induced fluorescence detection in capillary gel electrophoresis for accurate apolipoprotein E genotyping.
Human apolipoprotein E (apoE) is a product of a polymorphic gene. In the general population, it shows two major mutations, which lead to the appearance of three common alleles encoding for three protein isoforms. This polymorphism is important in the regulation of lipid metabolism. Accurate apoE phenotyping or genotyping has become essential in clinical laboratories, since the epsilon 4 allele has been associated with cardiovascular and Alzheimer's diseases. Endonuclease restriction isotyping, followed by slab gel electrophoresis, is a rapid and convenient method for the investigation of common apoE genotypes. However, during the large-scale apoE genotyping of the STANISLAS cohort, we were confronted with a partial lack of sensitivity and resolution power of this traditional method, which sometimes leads to the misclassification of the genotypes epsilon 2/2 and epsilon 3/2. We have overcome this difficulty by separating the restriction fragments with capillary gel electrophoresis linked to laser-induced fluorescence detection. The baseline resolution was 2 bp, and the sensitivity limit attainable was similar to that by radioactive detection. The distinction between the epsilon 3/2 and the epsilon 2/2 genotypes became unequivocal, even when only low amounts of DNA were available for amplification.
Schlenck
A
A
Centre de Médecine Préventive, Nancy, France.
Bohnet
K
K
Aguillon
D
D
Lafaurie
C
C
Siest
G
G
Visvikis
S
S
D000483:Alleles; D001057:Apolipoproteins E / Q000032:analysis / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005838:Genotype; D006801:Humans; D007834:Lasers; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity
D001057:Apolipoproteins E; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases