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Mol Cell Probes. 2005 Aug;19(4):290-7.

A whole-blood homogeneous assay for the multiplex detection of the factor V G1691A and the prothrombin G20210A mutations.

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  • 1Research Department, AGEN Biomedical Limited, P.O. Box 391, Acacia Ridge Q. 4110, Australia.


We describe an assay for the simultaneous detection in DNA or whole blood samples of the G1691A (Leiden) mutation in the factor V allele and the G20210A mutation in the prothrombin allele, both of which predispose humans to thrombosis. The assay uses a dual-probe quenching system in which one probe of a pair is labelled with a fluorophore and the adjacent probe is labelled with a fluorescence quencher. The primer partners were present in unequal amounts, and the genotypes were identified by melt curve analysis, which, together with the PCR, was performed in a Rotor-Gene centrifugal thermal cycler. The performance of the assay was assessed by consistency of melting temperature (Tm) values and height of melt curves from within- and between-run analyses. The stability of the PCR reagents and whole blood was assessed by determining Tm and peak height values after storage at -20, 4 degrees C or room temperature. Concentrations of the reaction reagents were finely adjusted to obtain consistent and clearly distinguishable melt curves that facilitated automatic assignment of 43 genotypes comprising wild-type and mutant alleles. The mean (SD) Tms and peak heights of 31 DNA samples assayed in a single run were 54.85 degrees C (0.07) and 2.33 U (0.15) for factor V and 53.69 degrees C (0.19) and 1.5 0U (0.08) for prothrombin. The Tms and melt peak heights for DNA from wild-type individuals (n=10) when assayed for between- and within-run variability showed SDs of <or=0.17 and <or=0.3 for Tm and peak height values, respectively. When whole blood was used as the sample, the assay performance was comparable to that of purified DNA samples, with whole blood assays (n=30) yielding mean (SD) Tms and peak heights of 53.74 degrees C (0.11) and 2.10 U (0.32) for factor V and 53.27 degrees C (0.12) and 2.15 U (0.18) for prothrombin. Reaction reagents stored for 12 months at room temperature produced satisfactory results, as did whole blood stored frozen for 2 years or in mineral oil at room temperature for at least 1 week. The features of the homogeneous PCR assay described in this article should facilitate the use of such assays in clinical laboratories.

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