GENOTYPING OF HUMAN APOLIPOPROTEIN-E ALLELES BY THE NEW QUALITATIVE, MICROPLATE-BASED CASSI-DETECTION ASSAY

A new qualitative PCR product detection assay called competitive ampli fied single mutation detection by selective probe hybridization immuno assay (CASSI) tvas developed for genotyping the most common apolipopro tein E (apoE) polymorphisms. Single target DNA strands immobilized usi ng biotin on streptavidin-coated microplates were hybridized in separa te wells with two distinct, 5'-fluorescein isothiocyanate (FITC)-label ed oligonucleotides, complementary to either the 112Arg or 158Arg enco ding site. With this assay, only correctly matched hybrids that form b etween probe and target DNA can be cleaved with the HhaI restriction e ndonuclease, lending to loss of probe label in corresponding wells. Ho wever, allele-specific, probe-target mismatches due to G-->T exchanges in the HhaI recognition sequences are not cleaved. After digestion, t he remaining microplate-adsorbed signal is measured colorimetrically b y using anti-FITC, Fab-horseradish peroxidase conjugates. Our results show maximum intensify was detected when the respective probe hybridiz ed incompletely to the target (i.e., no cleavage), and minimum signal was obtained when the probe matched the target completely (complete cl eavage); whereas, art intermediate signal was recorded at 50% compleme ntarity (i.e., heterozygote alleles). With this assay we could demonst rate a high prevalence of the apoE2 allele in patients suffering from coronary artery disease even though they displayed normal triglyceride and cholesterol levels. Corresponding results were obtained by CASSI compared with conventional restiction fragment-length polymorphism ana lysis.