Abstract:
BACKGROUND: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. METHODS: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. RESULTS: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). CONCLUSIONS: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.
journal_name
Clin Chem Lab Medjournal_title
Clinical chemistry and laboratory medicineauthors
Park HD,Park KU,Kim KW,Song J,Chang HE,Heo SR,Lee HJ,Kim JQdoi
10.1515/CCLM.2007.075subject
Has Abstractpub_date
2007-01-01 00:00:00pages
346-50issue
3eissn
1434-6621issn
1437-4331journal_volume
45pub_type
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