Optimization of Genotyping of the Common Apolipoprotein E Gene Alleles by Restriction Analysis

Abstract

Restriction isotyping is a convenient method to perform Apo E genotyping. However, some experimental conditions can affect the restriction isotyping prpcedure and can lead to results prone to misinterpretation. It was evaluated whether the concentration of teraplate DNA or different methods of DNA Isolation have an influence on restriction isotyping. Reproducible results were achieved with 0.05l ng genomic DNA in a 100 PCR mixture. Using less DNA resulted in the appearance of non-specific bands. This could lead to a misinterpretation of the band pattern. An influence of different DNA Isolation procedures on restriction isotyping could be excluded. The appearance of the band pattern is influenced by the intensity of the bands which depends on the size of the fragment and on the formation of heteroduplexes and homoduplexes in the case of a heterozygote apo E genotype. A detailed Interpretation of the band patteras obtained by restriction isotyping of the apo E gene is given with regard to the intensity of the bands following ethidium bromide staining of the gel and UV photography.