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  • Certification of a Reference Material consisting of purified plasmid DNA containing a fragment from the human prothrombin gene (G20210A mutant), IRMM/IFCC-492

Abstract

This report describes the production and certification of IRMM/IFCC-492, a reference material consisting of a mix of equal volumes of identical mass concentrations of two plasmid DNA (pDNA) containing either the wildtype or the G20210A mutant sequence of a 609 base pairs fragment for the human Factor II (prothrombin) gene (IRMM/IFCC-490 and IRMM/IFCC-491). This certified reference material (CRM) is intended to support the validation and the harmonisation of polymerase chain reaction (PCR)-based methods used for detection of the G20210A mutation in the prothrombin gene. The homogeneity and short-term stability of the batch was assessed by monitoring the melting temperature (Tm) of the product and the crossing point threshold (Cp) value obtained after PCR amplification using the LightCycler Factor II (prothrombin) G20210A kit and a LightCycler 1.5 instrument. The long-term stability was assessed using the cycle threshold value (Ct) obtained after real-time PCR amplification using specific primers and probe in combination with ABI Prism instruments.
The material is homogeneous and stable at -20 °C. The sequence of the insert in both plasmids (containing the wildtype sequence or the G20210A mutation) was confirmed by DNA sequence analysis. Although a second mutation (A->G) was found at position 20054 in IRMM/IFCC-491, its location did not influence the G20210A diagnostic protocol using the Factor II (prothrombin) G20210A kit. Each vial contains an indicative volume of 50 µL corresponding to approximately 1 ng of pDNA.

Additional information

Authors: GANCBERG D, European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel (BE);MARKI-ZAY J, European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel (BE);CORBISIER P, European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel (BE);SCHIMMEL H, European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel (BE);EMONS H, European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel (BE);KLEIN C, European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Ispra (IT)
Bibliographic Reference: EUR 22167 EN (2006), 33 pp. Free of charge
Availability: Available from European Commission, JRC Knowledge Management Unit, Ispra (IT) Tel: +39 033278 9893 Fax: +39 033278 5409 E-mail: jrc-publications-office@ec.europa.eu or Bruxelles (BE) Tel: +32 2 295 76 24 Fax: +32 2 299 63 22
ISBN: ISBN: 92-79-01632-6
Record Number: 200618679 / Last updated on: 2006-08-11
Category: PUBLICATION
Original language: en
Available languages: en