Simple and rapid method for extraction of DNA from fresh and cryopreserved clotted human blood.

DNA for use in polymerase chain reactions (PCR) and other molecular testing is most commonly isolated from anticoagulated blood. With the need to minimize blood volume used in laboratory testing, the sharing of samples among laboratories for a variety of different tests and extraction of DNA from blood clots that would otherwise be waste products is becoming increasingly important. Several methods for the isolation of DNA from clots have been described. Some of these methods provide DNA that is only suitable for PCR [1]. Other methods, although they provide high-quality genomic DNA, are cumbersome and tedious, involving either homogenization of the clot [2] or slicing it with a scalpel into small pieces [3, 4]. Homogenization probes aid tubes need very careful cleaning and chemical treatment between the samples to avoid cross-contamination. Clot slicing is very cumbersome and exposes laboratory personnel directly to blood-contaminated sharp instruments. Both approaches are impractical for preparation of large numbers of DNA samples, an important consideration when choosing a method for routine clinical or epidemiological use or intervention involving hundreds or even thousands of samples. We have developed a simple and rapid method for processing a large number of blood clots for isolation of high-quality DNA. Blood from healthy volunteers was collected into 10-mL serumseparator tubes and allowed to clot at room temperature. The clots were separated and either processed for DNA extraction on the same day or frozen before the extraction of DNA. The first step in the extraction of DNA from a blood clot involves mechanical breakage of the clots into small pieces. Everson et al. [2] used homogenization to break the clot, whereas Ta [3] and Siafakas et al. [4] used scalpels to slice the clot. Ta also attempted to homogenize the clots but stated that this procedure was difficult to optimize. We discovered that passing the blood clot through a nylon mesh (250 or 1000 p.m pores; Tetko, Monterey Park, CA) by using centrifugal force worked well for this homogenization. The mesh was attached to the top of a 50-mL conical-bottom polypropylene centrifuge tube and shaped into the form of a funnel, being held in place with a rubber band. The blood clot was poured into the mesh funnel and Parafilm#{174} (American Can Co., Neenah, WI) was wrapped around the top of the tube. The clot was then forced through the mesh funnel by centrifugation at 8000g for 10 mm. DNA from the sieved clot was then purified by using a commercial saltprecipitation method (Puregene Kit; Gentra Systems, Minneapolis, MN). Briefly, red blood cells were lysed with two washings of a hypotonic solution containing ammonium chloride, EDTA, and sodium bicarbonate. The white cells were then lysed by a sodium dodecyl sulfate solution, and the lysate was incubated with RNase to remove contaminating RNA. After protein precipitation with ammonium acetate, the DNA was precipitated with isopropanol and redissolved in a Tris-EDTA solution. The DNA was quantified and purity was determined from the absorbance ratio at 260/280 nm [5]. We compared the quantity and purity of DNA extracted from blood clots with 1000and 250-p.m mesh to mechanically break the clots. We found that the quantity of DNA was higher when 1000-jim mesh was used, compared with 250-jim mesh; however, the purity was lower as assessed by the A260/A280 ratio (quantity, mg/L whole blood: 30.8 ± 10.7 vs 21.7 ± 10.9; ratio: 1.56 ± 0.05 vs 1.73 ± 0.03; n = 10 and 6, respectively). We therefore used 250-j.Lm mesh to disrupt the blood clots in the remainder of our study. We then compared the quantity and purity of DNA extracted from blood obtained from 15 healthy volunteers using (a) EDTA-anticoagulated blood, (b) a clot that had been allowed to stand at room temperature <3 h, and (c) a clot that had been frozen for at least 1 week. The amount of DNA recovered from anticoagulated whole blood was greater than that collected from fresh or frozen clots (mean ± SD): 29 ± 13, 12 ± 9, and 12 ± 9 mg/L respectively. For a single 7-mL clot tube, the yield of DNA from fresh or frozen clots, although lower than that of anticoagulated blood, was ample for hundreds of PCR-based molecular tests. The A260/A280 ratio of DNA extracted from all samples was >1.7. The integrity of DNA was checked by agarose gel electrophoresis and all samples appeared intact. To test the DNA’s suitability for PCR testing, we also amplified the DNA with primers complementary to the cDNA sequence of the human cystathionine -synthase gene [6], which we were running routinely in our laboratory at the time; all DNA samples amplified without problems. In conclusion, the use of 2 50-p.m nylon mesh to mechanically break the blood clot followed by a routine method for extraction of DNA from blood is a simple, fast, inexpensive, and reliable procedure for obtaining acceptable quantities of high-quality DNA from large numbers of clotted blood specimens. Freezing of the clot had no measurable effect on the quantity or purity of the DNA extracted. We believe that this method will enable clinicians, researchers, and epidemiologists to use either fresh or frozen blood clots as a source of DNA in cases where serum is routinely obtained for other studies.