Quantitative assessment of apolipoprotein E genotypes by image analysis ofPCR-RFLP fragments

Apolipoprotein E (APOE) genotyping usually involves polymerase chain reacti on (PCR) and assessment of restriction fragment length polymorphism (RFLP) by gel electrophoresis. We made determination of HhaI restriction endonucle ase digestive patterns more objective and improved diagnostic accuracy with a quantitative approach using sensitive DNA stain (SYBR Green) and image a nalysis of gel patterns. For distinguishing true and partially-digested res triction fragments, band ratios were calculated for the staining intensity of gel patterns from 116 sample runs of 63 human blood specimens. Each of t hese specimens was independently genotyped for APOE by at least two (and mo st of them by three) different PCR-RFLP methods. Based on the distribution of band ratios, decision levels were established and used for developing a program for computer-aided interpretation of APOE genotypes (Microsoft Exce l software). Appropriateness of the decision levels for band ratios was val idated by APOE genotyping of additional 61 specimens. The approach describe d here is applicable to a variety of other molecular diagnostic techniques that are based on PCR-RFLP or sequence-specific signal amplifications. Publ ished by Elsevier Science B.V.