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Journal of Thrombosis and Thrombolysis

, Volume 16, Issue 3, pp 189–193 | Cite as

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

  • Antonio Angelini
  • Concetta Di Febbo
  • Giovanna Baccante
  • Marcello Di Nisio
  • Carmine Di Ilio
  • Franco Cuccurullo
  • Ettore Porreca
Article

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 μg/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 μg/ml; A260/A280 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 particulary suitable for a large scale screening for the diagnosis and prevention of the thrombotic risk.

venous thrombosis DNA preparation multiplex PCR alternative methods 

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Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Antonio Angelini
    • 1
  • Concetta Di Febbo
    • 1
  • Giovanna Baccante
    • 1
  • Marcello Di Nisio
    • 1
  • Carmine Di Ilio
    • 1
  • Franco Cuccurullo
    • 1
  • Ettore Porreca
    • 1
  1. 1.Centre of Excellence on Aging and Department of Medicine& Aging “G. D'Annunzio” University, School of MedicineChietiItaly

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