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A simple method for DNA isolation from clotted blood extricated rapidly from serum separator tubes.

Earlier assay methods required microgram quantities of DNA, but new high-throughput, highly sensitive assays allow genotyping of DNA samples at individual loci or throughout the genome with [less than or equal to]200 ng of genomic DNA per assay (1, 2) and have led to the exploration of nontraditional sources of DNA for research studies. The clinical pathology laboratory is a rich source of voluminous biochemical data for studies on the genetic basis of many human disorders or biochemical phenotypes, as long as they are conducted in a manner compliant with the Health Insurance Portability and Accountability Act with Institutional Review Board approval. One of the drawbacks of such an approach is that if a clinical pathology laboratory collects only serum for biochemical analysis, and the findings must then be correlated with genetic polymorphisms and mutations, it is often too late or cumbersome to acquire an additional blood sample to extract genomic DNA (3-4).

To resolve this issue without the further involvement of the patient, methods have been devised to recover DNA from clotted blood samples remaining in the serum-separation tubes after serum removal. Currently available methods, however, involve problematic and inconvenient fragmentation of the clotted blood before the removal of the sample from the tube (5). As a solid mass, the clot is difficult to manipulate at the bottom of the tube. More over, the separation gel obstructs the full extraction of the clot, thus leading to contamination of the sample with the separation gel and reduced yields of DNA. The fragmentation of the clot before its extraction also exposes the handler to the risk of direct contact with the blood (6). Because leukocytes are present throughout the clot, fragmentation of the entire clot is required to maximize their collection. By fragmenting the blood clot within the serum-separator tube, a significant amount of the sample is either lost or its quality compromised due to the mixing of the blood clot with the layer of separation gel covering it. We have developed a method that allows the complete and easy removal of the blood clot from a serum separation tube with minimal loss or contamination of the sample and minimal direct contact with the clot.

[FIGURE 1 OMITTED]

The primary innovation of this method is the extraction of the whole blood clot from the 10-mL serum-separator tube with minimal direct contact. This extraction was achieved by removal of the serum from the tube, leaving behind the serum-separator gel with the blood clot trapped below it, followed by centrifugation of the inverted serum-separator tube in a 50-mL polypropylene tube (ISC Bioexpress). During centrifugation at 10008 in a static-inclined rotor (20008 in a swing-bucket rotor), the lighter blood clot displaced the serum-separator gel and was repositioned at the accessible end of the tube with the separation gel layered below it (Fig. 1A; steps 1 and 2). A cone of 20-gauge steel mesh (The Home Depot) was created in the top of a 50-mL polypropylene tube by placing a 2-inch square of mesh, which had been sterilized by exposure to ultraviolet light in a tissue culture hood for 1 h, and depressing it into the 50-mL Falcon tube with a gloved thumb. This created a cone that fit snugly into the top of a 50-mL Falcon tube and had 4 flanges created by the 4 corners of the square mesh that anchored the mesh at the top of the tube when the cap was in place (Fig. 1A; step 3). The tube was uncorked and the entire blood clot easily transferred onto the cone with minimal direct contact. After the blood clot was placed into the mesh cone, the cap was applied tightly to immobilize the cone. The centrifugal force applied to the tube during the centrifugation process propelled the blood clot through the mesh, shearing it into a manageable mass. Unlike previous methods (6), this procedure eliminated the extensive manipulation of individual blood clots, allowing for simultaneous processing of a large number of blood clots. It also maximized blood clot recovery while minimizing exposure of the handler to potential blood-borne hazardous agents.

DNA extraction from the fragmented blood clot mass obtained from the shearing of the clot followed the standard procedures typically used for DNA extraction from whole blood. We added 20 mL erythrocyte lysis buffer to the fragmented clot in the 50-mL polypropylene tube, then vortex-mixed the tube briefly. The sample was then centrifuged at 20008 and the supernatant was discarded. A pale white leukocyte pellet was observed at the bottom of the 50-mL polypropylene tube, 11 mL cell lysis buffer was added, and the sample was vortex-mixed vigorously. We then added 1.2 mg proteinase K and 2 mL 10% sodium dodecyl sulfate to the sample, which was gently inverted to obtain a homogeneous mix. After incubation at 65 [degrees]C overnight, the sample was allowed to cool to room temperature before the addition of 4 mL protein precipitation solution. After centrifugation of the sample at 2000g, the supernatant was decanted into a fresh 50-mL polypropylene tube containing 100 [micro]g glycogen (Sigma-Aldrich) and precipitated with 12 mL isopropanol (Sigma-Aldrich). After incubation at room temperature for 15 min, the tube was centrifuged at 20008 to pellet the DNA and the supernatant was removed. The pellet was then washed in 12 mL 70% ethanol (Sigma-Aldrich) before the final centrifugation at 20008 to ensure that the DNA had formed a tight pellet at the bottom of the 50 mL polypropylene tube. The extracted DNA was dried by inverting the tube for 10 min at room temperature and then resuspended in 100 [micro]L of 1 x Tris-EDTA (TE) buffer (0.1 mol/L Tris-HCI; 0.01 mol/L EDTA; Sigma-Aldrich) before storage at -20 [degrees]C. We determined that 5 mol/L of ammonium chloride (Sigma-Aldrich) was the optimal protein precipitation solution. The composition of the erythrocyte lysis buffer (6) and cell lysis buffer (7) were as previously described.

The quality of the DNA was comparable to that extracted from whole blood directly (Fig. 1B). The total yield of DNA, as measured by NanoDrop (NanoDrop Technologies), from 7 samples processed on the same day as blood collection had a mean (SD) of 37.1 (33.4) [micro]g [range 13.3-107.8 [micro]g, 95% confidence interval (6, 68) [micro]g]. Because it is unlikely that samples will be processed as rapidly in a clinical setting, we identified 186 samples that had been stored at 4 [degrees]C for 1 month before DNA extraction according to the above protocol. These samples gave a mean (SD) DNA yield of 0.439 (0.470) [range 0.00194-2.905 [micro]g; 95% confidence interval (0.4, 0.5) [micro]g]. The quantity of DNA recovered from the samples therefore appears to be affected by the length of time between the collection of the blood sample and extraction of the DNA, making it important to extract DNA from the blood clot as soon as possible after collection. To test the quality of the DNA collected from the 1-month-old samples for future PCR-based analysis, a 572-bp region in the promoter region of the PAX9 gene was amplified (primer sequences and PCR conditions available on request) in 5 samples and a control DNA sample purified from whole blood using the Gents Puregene system (Gents Systems). The DNA samples obtained from the blood clot did not differ from the control DNA in their ability to serve as a template as compared with that of the control DNA (Fig. 1C).

We report a new method for the purification of DNA from clotted blood extracted from 10-mL serum-separator tubes that are commonly used in clinical testing. We have found this method to be an improvement over those previously reported because it: (a) minimizes the handling of the clot and the barrier imposed by the separation gel, (b) minimizes the possible contamination of the blood clot by both the separation gel and external factors, (c) maximizes the fragmentation of the clot, thereby enhancing the yield of DNA, (d) allows for the expedited processing of a large volume of samples, and (e) minimizes exposure of the handler to potential blood-borne pathogens.

S.S.F.W. and J.J.K. were supported by the Edmondson Summer Fellowship Program in the Department of Pathology at the University of Southern California. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 (RR10600-01, CA62528-01, RR1451401) from the NCRR, National Institutes of Health.

Previously published online at D01: 10.1373/clinchem.2006.078212

References

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(2.) Warrington JA, Shah NA, Chen X, Janis M, Liu C, Kondapalli S, et al. New developments in high-throughput resequencing and variation detection using high-density microarrays. Hum Mutat 2002;19:402-9.

(3.) Morabia A, Cayanis E, Costanza MC, Ross BM, Bernstein MS, Flaherty MS, et al. Association between lipoprotein lipase (LPL) gene and blood lipids: a common variant for a common trait? Genet Epidemiol 2003;24:309-21.

(4.) Kotowski IK, Pertsemlidis A, Luke A, Cooper RS, Vega GL, Cohen JC, et al. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol. Am J Hum Genet 2006;78:410-22.

(5.) Garg UC, Hanson N, Tsai MY, Eckfeldt JH. Simple and rapid method for extraction of DNA from fresh and cryopreserved clotted human blood. Clin Chem 1996;42:647-8.

(6.) Salazar LA, Hirata MH, Cavalli SA, Machado M0, Hirata RD. Optimized procedure for DNA isolation from fresh and cryopreserved clotted human blood useful in clinical molecular testing. Clin Chem 1998;44:1748-50.

(7.) Signer E, Kuenzle CC, Thomann PE, Hubscher U. DNA fingerprinting: improved DNA extraction from small blood samples. Nuc Acids Res 1988;16:7738.

Steven Se Fum Wong, [1] Jeffrey J. Kuei, [1] Naina Prasad, [1] Etsemaye Agonafer, [1] Gustavo A. Mendoza, [1] Trevor J. Pemberton, [1] and Pragna I. Patel [1,2] *

[1] Institute for Genetic Medicine and the [2] Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA

* Address correspondence to this author at: Institute for Genetic Medicine, University of Southern California, 2250 Alcazar Street, CSC-240, Los Angeles, CA 90033; fax 323-442-2764, e-mail pragna@usc.edu)
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Title Annotation:Technical Briefs
Author:Wong, Steven Se Fum; Kuei, Jeffrey J.; Prasad, Naina; Agonafer, Etsemaye; Mendoza, Gustavo A.; Pembe
Publication:Clinical Chemistry
Date:Mar 1, 2007
Words:1708
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