Optimized factor V gene mutation detection using buffy-coat direct PCR.

Activated protein C-resistance (APC-resistance) (6) is the most common inherited genetic disorder responsible for venous thrombosis (13). Over 90% of APC-resistances are caused by a single-base mutation, G/A 1691 in the factor V gene, also known as factor V Leiden. This mutation, changing Arg 506 to Gln, impedes the cleavage of factor Va by APC (3,7,14). APC-resistance is routinely determined by an activated partial thromboplastin time-based assay (6). Because this assay is often inadequate in cases of associated coagulation disorders (e.g., Lupus anticoagulant), diagnosis could be performed by DNA-based assays. Genotypic analysis is also proposed for individuals with borderline or low APC ratios. The combination of both phenotypic and genotypic profiles is more accurate in determining the associated increase in risk of thrombosis (5). For this genotypic analysis, detection of the G/A 1691 mutation is based on polymerase chain reaction (PCR) amplification of genomic DNA, followed by MnlI restriction enzyme digestion (3,15). Since purification of DNA is fastidious and time-consuming, several techniques have been developed for direct amplification of DNA from blood cells by PCR. Most of these reported methods require pretreatment of the blood samples, such as several washes (2,9, 10,12). To simplify the genetic diagnosis, we optimized direct PCR amplification using buffy coat. Because no DNA extraction was required, this method, derived from Mercier et al. (11), is faster and easier than the one described by Bertina et al. (3) for the detection of factor V Leiden. Fresh blood was collected into 0.129 M trisodium citrate anticoagulant (9/1 vol/vol) (Becton Dickinson, Meylan, France) and centrifuged at 2000× g for 15 min at room temperature. Plateletfree plasma was stored for further determination of phenotypic APC-resistance, and buffy coat was stored at -20°C until use. Optimization of the PCR protocol was performed according to the method of Cobb and Clarkson (4). Contrasting with more traditional optimization strategies including all possible combinations, this method, which uses the properties of orthogonal arrays, investigates the effects of a number of variables and the interaction among them in a single experiment containing a few reactions. Optimal concentrations of primers, dNTP and MgCl2, were thus determined (Table 1) in order to obtain an amplified product using a broad range of DNA concentrations from buffy coat. Our optimized conditions were: 5 μL of thawed buffy coat, 80.5 μL H2O and 10 μL Taq DNA Polymerase 10× buffer containing 1.5 mM MgCl2 (Appligene, Illkirch, France). The sample treatment was: 94°C, 3 min; 55°C, 3 min; 3 cycles (11). Then, 0.5 μM of each primer (PR6967, 5′-TGCCCAGTGCTTAACAAGACCA (3), V79100, 5′-CTTTGAAGGAAATGCCCCATTA) (8), 0.2 mM of each dNTP (Boehringer Mannheim, Meylan, France) and 2.5 U of Taq DNA Polymerase (Appligene) were added. After another denaturation step at 95°C for 4 min, the PCR conditions were: 91°C, 40 s; 55°C, 40 s; 72°C, 2 min; 36 cycles. This was followed by a final elongation step of 7 min at 72°C (3). A 220-bp amplified DNA fragment was electrophoresed on a 2% agarose or an 8% polyacrylamide gel and stained using ethidium bromide. Ten microliters of the PCR-amplified sample were directly digested overnight at 37°C by the MnlI restriction enzyme, 1 U in NEBuffer containing 1 mg/mL bovine serum albumin (BSA) (New England Biolabs, Beverly, MA, USA). Comparative studies were performed by PCR amplification of purified genomic DNA. This genomic DNA had been isolated from blood by proteinase K digestion followed by a phenol-chloroform extraction (1). Our direct PCR allowed for the amplification of the 220-bp DNA fragment of the factor V gene (14) containing the putative mutated site at position 1691 (Reference 3; Figure 1; Figure 2A; Figure 3). Using the method described by Cobb and Clarkson (4), optimal con-