| Popis: |
A technique to determine epsilon 2, epsilon 3 and epsilon 4 alleles expressed at the apolipoprotein E locus (apoE genotype) is described. The proposed method is convenient for detecting this polymorphism on capillary blood spots. Capillary blood is collected on absorbent paper allowing transmission by post and prolonged conservation of samples. Even when the amount of DNA is very small, double amplification by polymerase chain reaction (PCR) from a DNA fragment comprising the two polymorphic sites enables the length of the synthetized fragment to be measured the amplification of all samples to be verified, thus avoiding false interpretations resulting from a 51-base-pair fragment due to primer self-hybridization. The digestion of this fragment by Hha I restriction enzyme and electrophoresis of the digested products give an unambiguous diagnosis of the six most frequent (epsilon 2/epsilon 2, epsilon 3/epsilon 3 epsilon 4/epsilon 4, epsilon 2/epsilon 3, epsilon 2/epsilon 4, and epsilon 3/epsilon 4). Intended for genotype screening determinations, this technique is not convenient for all rare apoE variants, which must be determined by plasma isoelectrofocusing or genomic DNA sequencing. The technique may be done performed any time, even if the subject is not fasting. It avoids the difficulties of interpretation of the isoelectrophoretic patterns induced by poor conservation of the samples or the presence of sialylated isoforms of apoE or other contaminant proteins. The modest cost of the proposed technique allows determination of the apoE genotype in large series. |
