title: Identification of three genetic risk factors for venous thrombosis using a multiplex allele-specific PCR assay: comparison of conventional and new alternative methods for the preparation of DNA from clinical samples.
abstract: The demand for thrombophilia testing at the molecular level is increasing and with it the need for a simple and rapid and cost-saving procedure for the preparation of genomic DNA from whole blood samples. The aim of this paper is to compare the efficiency of two conventional commercial procedures (Genomic, Eurobio-Labtek, and Nucleospin, Macherey-Nagel) and two our alternative approaches (microwave irradiation and resin-binding method) for extraction DNA and their suitability and convenience for multiple sample preparation for simultaneous identification of the factor V Leiden, prothrombin 20210 and methylene tetrahydrofolate reductase (MTHFR) 677 variants by multiplex allele specific amplification (ASA-PCR). We have found that chemical-based kit (Genomic) produced higher DNA recovery (mean recovery 40 +/- 4.2 microg/ml; A260/A280 ratio 1,81 +/- 0.05) within 40 min., while the mini spin colum kit (Nucleospin Quickpure) obtained lower yield but the best DNA quality (mean recovery 25.7 +/- 2.3 microg/ml; A (260)/ A (280) ratio = 1,83 +/- 0.06) with fewer processing time (25 min). Costs of each extraction varied from 3.28 Euro for Genomic to 3.6 Euro for Nucleospin. Microwave radiation and resin-based method (GeneFizz) were single step/single tube procedures, that provided template DNA suitable for ASA-PCR assay, without any purification steps. The costs varied from 0.12 Euro for microwave to 1,23 Euro for resin based procedure. In conclusion, our alternative procedures were much faster (<15 min per extraction) and convenient (5.00-7.00 Euro per test) but equally sensitive compared to conventional DNA extraction methods. Moreover, these procedures are easily adaptable to the routine processing of high number of clinical samples and coupled with ASA-PCR assay result particularly suitable for a large scale screening for the diagnosis and prevention of the thrombotic risk.
authors.affiliation: Centre of Excellence on Aging and Department of Medicine & Aging G. D'Annunzio University, School of Medicine, Chieti, Italy. aangelini@unich.it
authors.forename: Antonio
authors.initials: A
authors.lastname: Angelini
authors.forename: Concetta
authors.initials: C
authors.lastname: Di Febbo
authors.forename: Giovanna
authors.initials: G
authors.lastname: Baccante
authors.forename: Marcello
authors.initials: M
authors.lastname: Di Nisio
authors.forename: Carmine
authors.initials: C
authors.lastname: Di Ilio
authors.forename: Franco
authors.initials: F
authors.lastname: Cuccurullo
authors.forename: Ettore
authors.initials: E
authors.lastname: Porreca
mesh_terms: D004247:DNA / Q000302:isolation & purification; D005165:Factor V; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D025202:Molecular Diagnostic Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011516:Prothrombin / Q000235:genetics; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D019851:Thrombophilia / Q000175:diagnosis / Q000235:genetics; D020246:Venous Thrombosis / Q000175:diagnosis* / Q000235:genetics*
chemical_list: D011933:Reagent Kits, Diagnostic; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin; D004247:DNA; D042965:Methylenetetrahydrofolate Reductase (NADPH2)