Genotyping of Human Apolipoprotein E Alleles by the New Qualitative, Microplate-Based CASSIDetection Assay. (July 1998)

• Record Type: Journal Article
• Authors: Köhler, T.; Rost, A.-K.; Purschwitz, K.; Vondran, S.; Remke, H.; Wagner, O.; Richter, V.
• Is Part Of: Biotechniques. Volume 25:Number 1(1998)
• Page Start: 80
• Page End: 85
• Publication Date: 1998-07
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/98251st03
• Languages: English
• ISSNs: 0736-6205

Abstract

A new qualitative PCR product detection assay called competitive amplified single mutation detection by selective probe hybridization immunoassay (CASSI) was developed for genotyping the most common apolipoprotein E (apoE) polymorphisms. Single target DNA strands immobilized using biotin on streptavidin-coated microplates were hybridized in separate wells with two distinct, 5′-fluorescein isothiocyanate (FITC)-labeled oligonucleotides, complementary to either the 112Arg or 158Arg encoding site. With this assay, only correctly matched hybrids that form between probe and target DNA can be cleaved with the Hha I restriction endonuclease, leading to loss of probe label in corresponding wells. However, allele-specific, probetarget mismatches due to G→T exchanges in the Hha I recognition sequences are not cleaved. After digestion, the remaining microplate- adsorbed signal is measured colorimetrically by using anti-FITC, Fabhorseradish peroxidase conjugates. Our results show maximum intensity was detected when the respective probe hybridized incompletely to the target (i.e., no cleavage), and minimum signal was obtained when the probe matched the target completely (complete cleavage); whereas, an intermediate signal was recorded at 50% complementarity (i.e., heterozygote alleles). With this assay, we could demonstrate 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 fragmentlength polymorphism analysis.