NDLI: Analytical evaluation of primer engineered multiplex polymerase chain reaction-restriction fragment length polymorphism for detection of factor V Leiden and prothrombin G20210A.

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Analytical evaluation of primer engineered multiplex polymerase chain reaction-restriction fragment length polymorphism for detection of factor V Leiden and prothrombin G20210A.

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Analytical evaluation of primer engineered multiplex polymerase chain reaction-restriction fragment length polymorphism for detection of factor V Leiden and prothrombin G20210A.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Huber, S. McMaster, K. J. Voelkerding, K. V.
Description	Country affiliation: United States Author Affiliation: Huber S ( Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison 53792, USA.)
Abstract	Factor V Leiden and prothrombin G20210A are clinically relevant genetic risk factors for venous thrombosis. Analysis for both mutations is increasingly being performed on patients exhibiting hypercoagulability. The goal of the current study was to evaluate the performance of primer-engineered multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the simultaneous detection of factor V Leiden and prothrombin G20210A. Primer-engineered multiplex PCR-RFLP methods for the detection of factor V Leiden and prothrombin G20210A from the medical literature were reviewed. A modified method was optimized in which both mutations generate HindIII RFLPs and the prothrombin amplicon contains an invariant HindIII recognition site to assess the completeness of endonuclease digestion. Digested amplification products were analyzed by agarose gel electrophoresis in a single gel lane and visualized by ethidium bromide. Primer-engineered multiplex PCR-RFLP was used to analyze 205 human genomic DNA samples whose factor V Leiden genotypes had been previously determined by MnlI PCR-RFLP. Complete concordance for factor V Leiden genotypes was observed between the two methods in the 205-sample cohort comprising 139 wild-type, 62 heterozygous mutant, and four homozygous mutant individuals. For prothrombin G20210A, primer-engineered multiplex PCR-RFLP identified 196 wild-type and nine heterozygous mutant individuals in the 205-sample cohort. To independently verify prothrombin genotypes, the nine heterozygous mutants and an additional 11 wild-type patient samples (representing 10% of patient samples) were subjected to DNA sequencing. Complete concordance was observed between DNA sequencing and primer-engineered multiplex PCR-RFLP results. In further validation, 123 of the DNA samples consisting of four heterozygous mutant and 119 wild type individuals were genotyped with the Invader Assay for Factor II (prothrombin G20210A). Results showed 100% concordance between the Invader Assay and primer-engineered multiplex PCR-RFLP. A primer-engineered multiplex PCR-RFLP based on single restriction endonuclease digestion has been evaluated and shown to simultaneously and accurately detect factor V Leiden and prothrombin G20210A mutations. The method is robust and readily adaptable to the clinical molecular diagnostic laboratory.
File Format	HTM / HTML
ISSN	15251578
e-ISSN	19437811
Journal	The Journal of Molecular Diagnostics
Issue Number	3
Volume Number	2
Language	English
Publisher	Elsevier
Publisher Date	2000-08-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Molecular Biology Factor V Genetics Mutation Polymerase Chain Reaction Polymorphism, Restriction Fragment Length Prothrombin Amino Acid Substitution Dna Mutational Analysis Dna Primers Deoxyribonuclease Hindiii Metabolism Electrophoresis, Agar Gel Genotype Reproducibility Of Results Sensitivity And Specificity Venous Thrombosis Research Support, Non-u.s. Gov't
Content Type	Text
Resource Type	Article
Subject	Molecular Medicine Pathology and Forensic Medicine

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