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Originally published online as doi:10.2353/jmoldx.2007.060035 on May 10, 2007

Published online before print May 10, 2007
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Journal of Molecular Diagnostics 2007, Vol. 9, No. 3
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2007.060035

Improved Real-Time Multiplex Polymerase Chain Reaction Detection of Methylenetetrahydrofolate Reductase (MTHFR) 677C>T and 1298A>C Polymorphisms Using Nearest Neighbor Model-Based Probe Design

Raghunath P. Agarwal*, Stephen M. Peters{dagger}, Manijeh Shemirani{ddagger} and Nicolas von Ahsen§

From the Department of Pathology, * Washington Hospital Center and Georgetown University Hospital, Washington, District of Columbia; the Departments of Pathology and Laboratory Medicine {dagger} and Laboratory Medicine, {ddagger} Georgetown University Hospital, Washington, District of Columbia; and the Department of Clinical Chemistry, § Georg-August University, Goettingen, Germany

The disorders of folate metabolism caused by methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms may lead to several disease states including coronary heart disease, venous thrombosis, and several types of cancer. We have developed a real-time multiplex single-tube polymerase chain reaction procedure on the LightCycler for the detection of the two most commonly occurring variants, 677C>T and 1298A>C, in the MTHFR gene. An improved probe design, based on the nearest neighbor model for nucleic acid-probe duplex stability, resulted in a better separation ({Delta}Tm ~ 10°C) of melting peaks of the wild-type and mutant alleles than that by the existing method ({Delta}Tm ~ 3°C) for specimens heterozygous for the 1298A>C polymorphism. Of the 333 blood specimens analyzed by this procedure, we did not find any samples that gave ambiguous results. The specimens with homozygous mutation for one polymorphism were of the wild type for the other variant. The assay was validated by the comparison of the genotyping results of 50 blood specimens from the LightCycler polymerase chain reaction with the conventional restriction fragment length polymorphism procedures. There was 100% concordance of the test results obtained by the two techniques. This assay is reliable, economical, and can be performed by less trained technologists compared with the procedure performed by the conventional restriction fragment length polymorphism technique.






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Copyright © 2007 by the American Society for Investigative Pathology and the Association for Molecular Pathology.