To the Editor:
The mutation in factor V (FV) G1691A, known as factor V Leiden,1 and the recently described genetic variation in the prothrombin (FII) gene G20210A2 are the two most prevalent known causes of inherited thrombophilia. Several polymerase chain reaction (PCR)-based methods have been described for the detection of each of them, separately. Although PCR is technically easy, it is rather expensive and time-consuming; the rate-limiting step is usually the DNA isolation. These arguments are important when considering the cost-benefit of large-scale prophylactic testing in individuals at risk. PCR amplification of FV Leiden from whole blood has been reported to be feasible and reliable.3,4 In addition, a method for the combined detection of both abnormalities by using purified DNA has been recently published.5 An ideal method would be the combination of both advantages, ie, a multiplex PCR for the simultaneous detection of both genetic abnormalities using whole blood as DNA source.
We report here a very rapid, simple, and cost-saving method for the genotypic diagnosis of both risk factors. This is achieved by multiplex PCR amplification of the involved region of both genes using whole blood as DNA source, followed by combined restriction digestion and agarose gel electrophoresis.
First, we compared the feasibility of multiplex amplifications on purified DNA using conditions for FII (Poort et al2 and R.M. Bertina, personal communication, December 1996) and FV1 with minor modifications. Despite the different conditions, both gene products were amplified using either of these two methods. However, the FII program gave stronger amplifications (not shown). FII amplifications were performed using 67 mmol/L Tris-HCl, pH 8.8; 16.6 mmol/L (NH4)2SO4; 6.7 mmol/L MgCl2; 10 mmol/L 2-mercaptoethanol; 100 μg/mL bovine serum albumin; 10% dimethylsulphoxide; 1.5 mmol/L of each dNTP; 2.5 U Taq DNA polymerase (Amplitaq; Perkin Elmer Cetus, Norwalk, CT); 500 ng of each FII; and 400 ng of each FV primer1,2 in a total volume of 50 μL. The sequence of the reverse FV primer (5′-CTTGAAGGAAATGCCCCATTA-3′) is different from that described.1 The FII program we use is as follows: After a denaturation step at 95°C for 4 minutes, thermal cycling was 1 minute of denaturing at 94°C, 1 minute of annealing at 53°C, and 2 minutes of extension at 67°C, with cycles repeated 32 times.
Subsequently, different amounts of whole blood (0.5, 1.0, 2.0, 5.0, and 10.0 μL) were assayed by adding ddH2O up to 20 μL before denaturation. The samples were denatured by heating the blood at 95°C for 5 minutes and then cooling at 30°C for 30 seconds, which was repeated three times. When using 1.0 or 2.0 μL of whole blood, both gene products were amplified using either the FII or the FV program. Again, the FII program resulted in stronger amplifications as compared with the FV program (Fig 1, lanes 1 and 2). Occasionally, low yields were obtained when using program FII. Therefore, we increased the number of cycles from 32 to 40. Because this resulted in consistent high yields (Fig 1, lane 3), we favor the use of 40 cycles for the multiplex amplification. Identical results were obtained when either fresh or frozen blood was used. When using half or one quarter of the amount of each of the primers, the yields were significantly lower (not shown).
For the genotype analysis, the 220-bp FV and 345-bp FII PCR products obtained by multiplex PCR were simultaneously digested by adding 6 UMnl I and 10 U HindIII (New England Biolabs, Beverley, MA) to 28 μL of PCR product, incubated for 2 hours at 37°C, and subsequently separated on agarose gel. The wild-type FII fragment contains two Mnl I sites but no HindIII sites, resulting in fragments of 15, 58, and 272 bp, whereas the 20210A (mutated) product contains two Mnl I sites and oneHindIII site, resulting in fragments of 15, 23, 58, and 249 bp after double digestion. The FV fragment contains no HindIII sites, resulting in Mnl I fragments of 37, 67, and 116 bp for the wild-type allele and 67 and 153 bp for the mutated allele. Consequently, when both PCR products of a patient double heterozygote for FII and FV are digested, fragments of 272, 249, 153, and 116 bp are detected (Fig 1, lanes 9 and 11).
In conclusion, the presented multiplex amplification on 2 μL of whole blood followed by a combined restriction digest of the obtained PCR products offers a very rapid, feasible, and cost-saving method for large-scale FII and FV genotype analysis.