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

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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 (Tm) was 62.8 °C, whereas a sample with the TGC/TGC sequence had a Tm of 54.7°C. A similar fluorescence pattern appeared with a different Tm at 66.9°C (CGC/CGC) and 59.7°C (TGC/TGC) for codon 158 of APOE. For the ChAT polymorphism, the melting temperature (61.4°C) of the G allele was higher than that of the A allele (54.7°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.

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