Optimized Factor V Gene Mutation Detection Using ... - BioTechniques

Optimized Factor V Gene Mutation Detection Using Buffy-Coat Direct PCR BioTechniques 22:837-841 (May 1997)

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-resisVol. 22, No. 5 (1997)

tance, 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 conCircle Reader Service No. 154

Benchmarks Table 1. Optimized Protocol for the Buffy-Coat Direct PCR of Factor V Leiden

Lane in Figure 1

Figure 1. Optimized protocol for the buffycoat direct PCR of factor V Leiden. Protocol was optimized according to the method of Cobb and Clarkson (4). Optimal conditions were chosen according to lane 9. MW: molecular weight marker (PBR322/HaeIII). Amplified DNA was electrophoresed on a 2% agarose gel.

centration of each component was determined (Table 1 and Figure 1, lane 9). Based on these optimal conditions, the results obtained were identical using either the optimized direct PCR (Figure 2A, lane 1) or purified DNA in the reaction (Figure 2A, lanes 2 and 3). Moreover, the amplified DNA fragment was directly suitable for MnlI restriction enzyme analysis (Figure 2B). Since the amount of DNA in buffy coat varies widely from one patient to

1 2 3 4 5 6 7 8 9

Buffy Coat (µL) 1 1 1 2 2 2 5 5 5

Primers (µM)

dNTP (mM)

MgCl2 (mM)

0.1 0.25 0.5 0.1 0.25 0.5 0.1 0.25 0.5

0.05 0.2 0.4 0.2 0.4 0.05 0.4 0.05 0.2

1.5 3 4.5 4.5 1.5 3 3 4.5 1.5

Protocol was optimized according to the method of Cobb and Clarkson (4). The table shows orthogonal array of conditions tested. Optimal conditions were chosen according to lane 9 in Figure 1.

another, our optimized protocol was tested with various amounts of buffy coat from one sample. The protocol allowed for a direct amplification from 1 µL up to 10 µL of buffy coat (Figure 3A) and an efficient enzymatic digestion (data not shown). In our experience, 5 µL of thawed buffy coat yielded the best results, without inhibiting the Taq DNA polymerase previously described (11). Moreover, similar amplification products were obtained after

Figure 2. (A) Comparison of buffy-coat direct PCR and PCR-amplification of purified DNA. MW: molecular weight marker (PBR322/HaeIII); lane 1: direct PCR from 5 µL of thawed buffy coat using optimized conditions; lanes 2 and 3: PCR from purified DNA, 130 ng (lane 2) and 325 ng (lane 3). (B) Amplified fragments digested by MnlI. 5 µL of thawed buffy coat were amplified according to our optimized protocol and then digested by MnlI as described above. Normal factor V allele is cleaved into fragments of 37 (not visualized), 67 and 116 bp. Digestion of the factor V Leiden allele results in fragments of 67 and 153 bp (8). MW: molecular weight marker (PBR322/HaeIII); lane 1: negative control; lane 2: normal patient; lane 3: heterozygous for factor V Leiden; lane 4: homozygous for factor V Leiden. Amplified DNA and digested fragments were electrophoresed on an 8% polyacrylamide gel. 840 BioTechniques

optimized PCR amplification of 5 µL of buffy coat from different patients (data not shown). Several parameters could explain this difference with the original method of Mercier et al. (11): (i) the buffy-coat preparation contains much less inhibiting hemoglobin per µL than does whole blood; compared with the original method, this could explain the ability to amplify greater volumes of blood containing DNA; (ii) the Taq DNA polymerase and primers were different; this could affect the efficiency of the amplification; (iii) our Taq DNA polymerase buffer contained 0.1% of Triton X-100; and (iv) a 4min denaturation step was added just before the amplification cycles; this additional step could enhance the removal

Figure 3. Direct PCR for various amounts of buffy coat. Lanes 2–6: using our optimized protocol, direct PCR (see text) was performed for 1 (lane 2); 2 (lane 3); 5 (lane 4); 10 (lane 5) and 20 µL (lane 6) of thawed buffy coat. MW: molecular weight marker (PBR322/HaeIII); lane 1: negative control. Amplified DNA was electrophoresed on 8% polyacrylamide gel. Vol. 22, No. 5 (1997)

of potential inhibitors such as hemoglobin. This buffy-coat direct PCR can be performed on either citrated or EDTAanticoagulated blood. Citrated blood was of particular interest since the same blood sample could be used for both plasma phenotypic and genotypic analyses of APC-resistance. This rapid buffy-coat direct PCR allowed for amplification using a simpler protocol than that of Bertina et al. (3). It is a very simple procedure because it requires neither DNA extraction nor pre-isolation of white blood cells, and it is well-suited for routine diagnostic work. REFERENCES 1.Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl. 1992. Preparation and analysis of data, p.2.2.1-2.2.2. Current Protocols in Molecular Biology. John Wiley & Sons, New York. 2.Balnaves, M.E., S. Nasioulas, H.H.M. Dahl and S. Forrest. 1991. Direct PCR from CVS and blood lysates for detection of cystic fibrosis and Duchenne muscular dystrophy deletions. Nucleic Acids Res. 19:1155. 3.Bertina, R.M., B.P.C. Koeleman, T. Koster, F.R. Rosendaal, R.J. Dirven, H. de Ronde, P.A. van der Veiden and P.H. Reitsma. 1994. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 369:64-67. 4.Cobb, B.D. and J.M. Clarkson. 1994. A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods. Nucleic Acids Res. 22:3801-3805. 5.Dahlbäck, B. 1995. Resistance to activated protein C, the Arg506 to Gln mutation in the factor V gene, and venous thrombosis. Thromb. Haemost. 73:739-742. 6.Dahlbäck, B., M. Carlsson and P.J. Svensson. 1993. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc. Natl. Acad. Sci. USA 90:1004-1008. 7.Greengard, J.S., X. Sun, X. Xu, J.A. Fernadez, J.H. Griffin and B. Evatt. 1994. Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 343:1362-1363. 8.Koeleman B.P.C., P.H. Reitsma, C.F. Allaart and R.M. Bertina. 1994. Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficient families. Blood 84:1031-1035. 9.Lewin, H.A. and J.A. Stewart-Haynes. 1992. A simple method for DNA extraction from leukocytes for use in PCR. BioTechniques 13:522-524. 10.McHale, R.H., P.M. Stapleton and P.L. Bergquist. 1991. Rapid preparation of blood and tissue samples for polymerase chain reacVol. 22, No. 5 (1997)

tion. BioTechniques 10:20-23. 11.Mercier, B., C. Gaucher, O. Feugeas and C. Mazurier. 1990. Direct PCR from whole blood, without DNA extraction. Nucleic Acids Res. 18:5908. 12.Nordvåg, B.-Y., G. Husby and M. Raafat El-Gewely. 1992. Direct PCR of washed blood cells. BioTechniques 12:490-493. 13.Svensson, P.J. and B. Dahlbäck. 1994. Resistance to activated protein C as a basis for venous thrombosis. N. Engl. J. Med. 330:517522. 14.Zöller, B. and B. Dahlbäck. 1994. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 343:1536-1538. 15.Zöller, B., P.J. Svensson, X. He and B. Dahlbäck. 1994. Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J. Clin. Invest. 94:25212524.

We thank A. Besson and C. Coudoux for technical assistance. Address correspondence to Gilles Pernod, Laboratoire d’Hématologie, Hopital A Michalon, CHU de Grenoble, BP 217, 38043 Grenoble Cédex 9, France. Received 14 February 1996; accepted 11 November 1996.

Gilles Pernod, Pascal Mossuz and Benoit Polack CHU de Grenoble Grenoble, France

Spectrophotometric Determination of Oxidative Metabolism BioTechniques 22:841-844 (May 1997)

Oxidative metabolism is a good indicator of the activation state of several cell types including macrophage(s) (Mφ), eosinophils and B cells (1,2,5,6). This indicator has been successfully used to monitor the activation state of Mφ and remains an important technique for experimental assessment of this complex process (2,7). Traditionally, oxidative metabolism has been assayed by the conversion of nitroblue tetrazolium (NBT) to blue/black formazan deposits in the target cells and the subsequent microscopic examina-

tion and enumeration of cells containing these deposits (2,3,4). This method is very labor-intensive and potentially subject to investigator bias. We have developed a method for examining this conversion spectrophotometrically, thereby eliminating observer subjectivity and allowing a more accurate determination in a fraction of the time. Initial experiments were conducted to compare the spectrophotometric assay with the traditional microscopic method. Mφ are plated at three different concentrations in a 96-well plate and examined both microscopically and spectrophotometrically. The procedure is carried out under sterile conditions by plating cells at 1–5 × 104 cells per well in 160 µL and adding 40 µL of NBT to each well. NBT is purchased from Sigma Chemical (St. Louis, MO, USA) as a lyophilized product in the presence of phosphate buffer and NaCl. It is resuspended in sterile H2O at 2.0 mg/mL. The unconverted NBT has an absorbance spectrum that peaks at 385 nm and falls to zero after 490 nm (Figure 1A). Once converted to formazan by the addition of NaOH, the absorbance spectrum varies only marginally, exhibiting a peak absorbance at 655 nm, well beyond the peak for the unconverted form (Figure 1B). Thus, no assay interference is demonstrated at wavelengths 490–700 nm. Additionally, since we artificially converted the NBT to formazan through the addition of NaOH to determine its peak absorbance, we examined the effect of altering pH on the absorbance at 655 nm. The changes in optical density (OD)655 did not vary as a function of pH changes within the range of pH 2.97–11.66 (data not shown). Likewise, the contribution of the cells themselves to the absorbance at 655 nm at each of the cell numbers examined was negligible (data not shown). Following the NBT addition, the plate is shaken gently for 1 min and incubated for 24 h in a humidified chamber at 37°C and 7% CO2. After 24 h, microscopic examination is performed using a Diaphot-TMD Phase Contrast Microscope (Nikon, Tokyo, Japan) at 100× final magnification, and the number of positive cells per field is determined. The plates are then shaken vigorously for 5 s, read on a SPECTRAmax 250 Plate Reader BioTechniques 841