Search

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 HhaI restriction endonuclease, leading to loss of probe label in corresponding wells. However, allele-specific, probe-target mismatches due to G-->T exchanges in the HhaI recognition sequences are not cleaved. After digestion, the remaining microplate-adsorbed signal is measured colorimetrically by using anti-FITC, Fab-horseradish 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 restriction fragment-length polymorphism analysis.

DNA fragments used as standards in competitive PCR were precisely calibrated using HPLC and commercially available DNA molecular mass markers. The accuracy of calibration was reflected by data that differed by only 2% from the mean when two independently purified and calibrated competitor preparations were compared. Highly dilute competitor solutions were stable at -20 degrees C for up to 1 year in the presence of carrier HindIII-digested lambda DNA, but progressive loss of competitor DNA with increasing storage time was observed when carrier DNA was omitted from the solution. Applying 0.2 U uracil-DNA glycosylase (UDG) per assay of remaining temperature-stable activity did not effect the ratios of synthesized products. This study describes quality management in PCR quantitation that is useful for the measurement of multidrug resistance-associated protein (MRP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts.