Document Detail


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.
MedLine Citation:
PMID:  17591934     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 (DeltaTm approximately 10 degrees C) of melting peaks of the wild-type and mutant alleles than that by the existing method (DeltaTm approximately 3 degrees 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.
Authors:
Raghunath P Agarwal; Stephen M Peters; Manijeh Shemirani; Nicolas von Ahsen
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Publication Detail:
Type:  Comparative Study; Evaluation Studies; Journal Article    
Journal Detail:
Title:  The Journal of molecular diagnostics : JMD     Volume:  9     ISSN:  1525-1578     ISO Abbreviation:  J Mol Diagn     Publication Date:  2007 Jul 
Date Detail:
Created Date:  2007-06-26     Completed Date:  2007-09-19     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  100893612     Medline TA:  J Mol Diagn     Country:  United States    
Other Details:
Languages:  eng     Pagination:  345-50     Citation Subset:  IM    
Affiliation:
Department of Pathology, Washington Hospital Center, NW, Washington, DC 20010, USA. rpagarwal@cox.net
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MeSH Terms
Descriptor/Qualifier:
Algorithms*
Base Sequence
DNA Mutational Analysis / methods*
Genotype
Humans
Methylenetetrahydrofolate Reductase (NADPH2) / genetics*
Models, Genetic
Molecular Sequence Data
Oligonucleotide Probes / chemical synthesis*,  chemistry
Polymorphism, Restriction Fragment Length
Polymorphism, Single Nucleotide*
Reverse Transcriptase Polymerase Chain Reaction / methods*
Sequence Homology, Nucleic Acid*
Transition Temperature
Chemical
Reg. No./Substance:
0/Oligonucleotide Probes; EC 1.5.1.20/Methylenetetrahydrofolate Reductase (NADPH2)
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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