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; 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 particulary suitable for a large scale screening for the diagnosis and prevention of the thrombotic risk.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Bertina RM, Koeleman BPC, Koster T, et al. Mutation in blood coagulation factor V associated with resistence to activated protein C. Nature 1994;369:64-67.
Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3_traslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698.
Arruda VR, von Zuben PM, Chiaparini LC, Annichino-Bizzacchi JM, Costa FF. The mutation ala677val in the methylene tetrahydrofolate reductase gene: A risk factor for arterial disease and venous thrombosis. Throm Haemost 1997;77:818-821.
Rosendaal FR. Risk factors for venous thrombotic disease. Thromb Haemost 1999;82:610-619.
Bertina RM. Genetic approach to thrombophilia. Thromb Haemost 2001;86:92-103.
Bottema CD, Sommers SS. PCR amplification of specific alleles: Rapid detection of known mutations and polymorphisms. Mutat Res 1993;288:93-102.
Bottema CD, Sarkar G, Cassadfy JD, Ii S, Dutton CM, Sommer SS. Polymerase chain reaction amplification of specific alleles: A general method of detection of mutations, polymorphisms, and haplotypes. Methods Enzymol 1993;218:388-402.
Sambrook JE, Fritsch F, Maniatis T. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor, 1989, vol. 1-3.
Hessner MJ, Luhm RA, Pearson SL, Endean DJ, Friedman KD, Montgomery RR. Prevalence of prothrombin G20210A, factor V G1691A (Leiden), and methylenetetrahydrofolate reductase (MTHFR) C677T in seven different populations determinated by multiplex allele-specific PCR. Thromb Haemost 1999;81:733-738.
Hezard N, Cornillet-Lefebvre P, Gillot L, Potron G, Nguyen P. Multiplex ASA-PCR for a simultaneously determination of factor V Leiden gene, G ? A 20210 prothrombin gene and C?T 677 MTHFR gene mutations. Thromb Haemost 1998;79:1054-1055.
Pecheniuk NM, Marsh NA, Walsh TP. Multiple analysis of three common genetic alterations associated with thrombophilia. Blood Coagul Fibrinolysis 2000;11:183-189.
Ohhara M, Kurosu Y, Esumi M. Direct PCR of whole blood and hair shafts by microwave treatment. BioTechniques 1994;17:726.
Banerjee SK, Makdisi WF, Weston AP, Michell SM, Campbell DR. Microwave-based DNA extraction from paraffin-embedded tissue for PCR amplification. BioTechniques 1995;18:768-772.
Angelini A, Di Febbo C, Rullo A, Di Ilio C, Cuccurullo F, Porreca E. New method for the extraction of DNA from white blood cells for the detection of common genetic variants associated with thrombophilia. Pathophysiol Haemost Thromb 2002;32:180-183.
About this article
Cite this article
Angelini, A., Di Febbo, C., Baccante, G. et al. 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. J Thromb Thrombolysis 16, 189–193 (2003). https://doi.org/10.1023/B:THRO.0000024057.37588.2a
- venous thrombosis
- DNA preparation
- multiplex PCR
- alternative methods