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

  • Hyung-Doo Park , Kyoung Un Park , Ki-Woong Kim , Junghan Song , Ho Eun Chang , Se Ran Heo , Hyun Jung Lee and Jin Q. Kim
Published/Copyright: March 1, 2007
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Volume 45 Issue 3

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 (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).

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.

Clin Chem Lab Med 2007;45:346–50.

Keywords: Alzheimer's disease; apolipoprotein E (APOE); choline acetyltransferase (ChAT); polymorphism; real-time multiplex PCR
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Corresponding author: Kyoung Un Park, MD, PhD, Department of Laboratory Medicine, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, Korea Phone: +82-31-7877692, Fax: +82-31-7874015,

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Published Online: 2007-03-01
Published in Print: 2007-03-01

©2007 by Walter de Gruyter Berlin New York

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https://doi.org/10.1515/CCLM.2007.075
Keywords for this article
Alzheimer's disease; apolipoprotein E (APOE); choline acetyltransferase (ChAT); polymorphism; real-time multiplex PCR

Articles in the same Issue

  1. From human genetic variations to prediction of risks and responses to drugs and the environment
  2. Nutrigenomics – 2006 update
  3. How to comprehensively analyse proteins and how this influences nutritional research
  4. Genotypes, obesity and type 2 diabetes – can genetic information motivate weight loss? A review
  5. The Gene-Diet Attica Investigation on childhood obesity (GENDAI): overview of the study design
  6. Polymorphisms in the APOA1/C3/A4/A5 gene cluster and cholesterol responsiveness to dietary change
  7. Nutri-epigenomics: lifelong remodelling of our epigenomes by nutritional and metabolic factors and beyond
  8. Emerging role of cathepsin S in obesity and its associated diseases
  9. Association analysis of hepatitis virus B infection with haplotypes of the TBX21 gene promoter region in the Chinese population
  10. Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality
  11. Real-time multiplex PCR assay for genotyping of three apolipoprotein E alleles and two choline acetyltransferase alleles with three hybridization probes
  12. Immunomagnetic CD45 depletion does not improve cytokeratin 20 RT-PCR in colorectal cancer
  13. Analysis of the components of hypertransaminasemia after liver resection
  14. Fine characterization of mitral valve glycosaminoglycans and their modification with degenerative disease
  15. Oxidative stress evaluated using an automated method for hydroperoxide estimation in patients with coronary artery disease
  16. Secretory phospholipase A2 activity and release kinetics of vascular tissue remodelling biomarkers after coronary artery bypass grafting with and without cardiopulmonary bypass
  17. Clustered components of the metabolic syndrome and platelet counts in Japanese females
  18. International Standard for serum vitamin B12 and serum folate: international collaborative study to evaluate a batch of lyophilised serum for B12 and folate content
  19. Multicentre physiological reference intervals for serum concentrations of immunoglobulins A, G and M, complement C3c and C4 measured with Tina-Quant® reagents systems
  20. In vivo and in vitro allergy diagnostics: it's time to reappraise the costs
  21. Experience with post-market surveillance of in-vitro diagnostic medical devices for lay use in Germany
  22. Evaluation of the high-sensitivity, full-range Olympus CRP OSR6199 application on the Olympus AU640®
  23. How to improve the teaching of urine microscopy
  24. In vitro determination of allergen-specific serum IgE. Comparative analysis of three methods
  25. Efficacy of a new blocker against anti-ruthenium antibody interference in the Elecsys free triiodothyronine assay
  26. Clinical indications for plasma protein assays: transthyretin (prealbumin) in inflammation and malnutrition: International Federation of Clinical Chemistry and Laboratory Medicine (IFCC): IFCC Scientific Division Committee on Plasma Proteins (C-PP)
  27. Meeting Report: From human genetic variations to prediction of risks and responses to drugs and the environment
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