Real-time multiplex PCR assay for genotyping of three apolipoprotein E alleles and two choline acetyltransferase alleles with three hybridization probes

Abstract

Apolipoprotein E (APOE) and choline acetyltransferase (ChAT) have been suggested as candidate genes for determining the risk of late-onset Alzheimer's disease. The aim of this study was to simultaneously detect polymorphisms in codons 112 and 158 of APOE and codon 2 of ChAT by hybridization probe multiplexing. The decrease in fluorescence emitted by LC-Red 610, LC-Red 640, and LC-Red 705 channels was quantified during a gradual temperature increase after amplification. The melting curves were converted to melting peaks by plotting the negative derivative of the fluorescence with respect to temperature (-dF/dT) as a function of temperature. A single pair of hybridization probes and PCR restriction fragment length polymorphism (RFLP) were used to confirm the genotyping of APOE and ChAT, respectively, in 183 subjects. When a homozygous sample with the CGC/CGC sequence in codon 112 of APOE was analyzed, the mean sequence-specific melting point (T(m)) was 62.8 degrees C, whereas a sample with the TGC/TGC sequence had a T(m) of 54.7 degrees C. A similar fluorescence pattern appeared with a different T(m) at 66.9 degrees C (CGC/CGC) and 59.7 degrees C (TGC/TGC) for codon 158 of APOE. For the ChAT polymorphism, the melting temperature (61.4 degrees C) of the G allele was higher than that of the A allele (54.7 degrees C). This real-time multiplex PCR technique can be carried out in a single tube and can differentiate between the three polymorphic sites of the two genes associated with Alzheimer's disease.