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Easy detection of 5,10-methylenetetrahydrofolate reductase 1298A/C genotype by mutagenically separated PCR assay

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Published/Copyright: May 22, 2008
Clinical Chemistry and Laboratory Medicine
From the journal Clinical Chemistry and Laboratory Medicine Volume 46 Issue 7

Abstract

Background: 5,10-Methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism. Previous studies have suggested an association between the MTHFR 1298A/C polymorphism and several diseases, such as cardiovascular and psychiatric diseases, neural tube defects, diabetes, and cancer. Currently, either PCR-restriction fragment length polymorphism (RFLP) technique or real-time PCR using Taqman assay are used to determine the MTHFR 1298 genotype.

Methods: We developed a simple and efficient approach that employs mutagenically separated PCR to genotype MTHFR 1298A/C polymorphism. Two forward mutagenic allele-specific primers of different lengths for MTHFR 1298A/C were paired with the same reverse primer in a one-tube assay to genotype 20 genomic DNA samples.

Results: Electrophoresis on 2.5% agarose gel showed two allele-specific fragments, a 113-bp A allele-specific and a 93-bp C allele-specific PCR product. The results were confirmed by the conventional PCR-RFLP method.

Conclusions: We conclude that mutagenically separated PCR could be used as an alternative simple, reliable, and cost-effective method for the genotyping of MTHFR 1298A/C polymorphism.

Clin Chem Lab Med 2008;46:987–9.

Keywords: genotyping; MTHFR; mutagenically separated PCR; single nucleotide polymorphism
Corresponding author: F. Naghibalhossaini, Department of Biochemistry, Shiraz University of Medical Sciences, School of Medicine, Zand Street, Shiraz 71345, Iran Phone/Fax: +98-711-2303029,
Received: 2008-1-25
Accepted: 2008-2-26
Published Online: 2008-05-22
Published in Print: 2008-07-01

©2008 by Walter de Gruyter Berlin New York

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https://doi.org/10.1515/CCLM.2008.184
Keywords for this article
genotyping; MTHFR; mutagenically separated PCR; single nucleotide polymorphism

Articles in the same Issue

  1. Editorial
  2. CCLM: Evolving to meet the needs of today's laboratory professionals and scientists
  3. Bacterial detection and blood product contamination
  4. Editorial: Rapid diagnostic tests to detect pathogenic microorganisms
  5. Reviews
  6. Rapid methods for diagnosis of bloodstream infections
  7. Validation criteria for nucleic acid amplification techniques for bacterial infections
  8. Incidence of bacterial transmission and transfusion reactions by blood components
  9. Strategies of bacteria screening in cellular blood components
  10. Methods for the detection of bacterial contamination in blood products
  11. Bacteria detection by flow cytometry
  12. Rapid screening by real-time 16S rDNA PCR for bacterial contamination of blood products
  13. Microbial safety of cell based medicinal products – what can we learn from cellular blood components?
  14. Genetics and Molecular Diagnostics
  15. Screening for genetic heterogeneity in the interferon sensitivity determining region of the hepatitis C virus genome by polymerase chain reaction with melting curve analysis
  16. Thiopurine S-methyltransferase (TPMT) pharmacogenetics: three new mutations and haplotype analysis in the Estonian population
  17. Genetic testing for adult-type hypolactasia in Italian families
  18. Improved real-time detection of the H63D and S65C mutations associated with hereditary hemochromatosis using a SimpleProbe assay format
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  21. Mediterranean diet and plasma concentration of inflammatory markers in old and very old subjects in the ZINCAGE population study
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  29. An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA
  30. A new general regression-based approach for method comparison studies
  31. Letters to the Editor
  32. Procalcitonin values in preeclamptic women are related to severity of disease
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  35. Lack of association of GH T1663A variation and IGFBP3–202A/C polymorphism with risk of ischemic stroke in Chinese
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