BACKGROUND: After clinical laboratory tests have been performed, it can be difficult to obtain DNA without further patient involvement. Although the blood clot remaining within the serum-separation tube after serum collection is a source of DNA, recovery of the clot from the tube is a significant challenge. METHOD: We devised a method to efficiently remove clotted blood from the serum-separation gel and extract DNA from clotted whole blood samples, obtaining maximum yield of the DNA without DNA contamination by the separation gel. The method involved centrifugation of the sample in the inverted original 10-mL collection tube to displace the separation gel for easy isolation of the blood clot and shearing of the blood clot by centrifugation through a 20-gauge wire mesh cone at 2000 g in a swinging-bucket rotor. After erythrocyte lysis and proteinase-K digestion of the fragmented clot, DNA was precipitated with isopropanol in the presence of glycogen. RESULTS: The mean amount of DNA obtained from a 4-mL clotted blood sample prepared by this method was 37.1 microg for clots processed soon after collection, with a reduction to 0.439 microg for clots stored for 1 month before extraction. The quality of the DNA was comparable to that extracted directly from whole blood, and it was found to be suitable for PCR-mediated analysis. CONCLUSION: We have formulated a method that overcomes the difficulties of safely extricating a blood clot from serum-separation tubes, allowing rapid DNA extraction for the purposes of genetic investigation.
BACKGROUND: After clinical laboratory tests have been performed, it can be difficult to obtain DNA without further patient involvement. Although the blood clot remaining within the serum-separation tube after serum collection is a source of DNA, recovery of the clot from the tube is a significant challenge. METHOD: We devised a method to efficiently remove clotted blood from the serum-separation gel and extract DNA from clotted whole blood samples, obtaining maximum yield of the DNA without DNA contamination by the separation gel. The method involved centrifugation of the sample in the inverted original 10-mL collection tube to displace the separation gel for easy isolation of the blood clot and shearing of the blood clot by centrifugation through a 20-gauge wire mesh cone at 2000 g in a swinging-bucket rotor. After erythrocyte lysis and proteinase-K digestion of the fragmented clot, DNA was precipitated with isopropanol in the presence of glycogen. RESULTS: The mean amount of DNA obtained from a 4-mL clotted blood sample prepared by this method was 37.1 microg for clots processed soon after collection, with a reduction to 0.439 microg for clots stored for 1 month before extraction. The quality of the DNA was comparable to that extracted directly from whole blood, and it was found to be suitable for PCR-mediated analysis. CONCLUSION: We have formulated a method that overcomes the difficulties of safely extricating a blood clot from serum-separation tubes, allowing rapid DNA extraction for the purposes of genetic investigation.
Authors: Mitchell H Gail; Tim Sheehy; Mark Cosentino; David Pee; Norma A Diaz-Mayoral; Montserrat Garcia-Closas; Neil E Caporaso; Karen Pitt; Regina G Ziegler Journal: Am J Epidemiol Date: 2013-07-14 Impact factor: 4.897
Authors: Simon Kaja; Anna A Shah; Shamim A Haji; Krishna B Patel; Yuliya Naumchuk; Alexander Zabaneh; Bryan C Gerdes; Nancy Kunjukunju; Nelson R Sabates; Michael A Cassell; Ron K Lord; Kevin P Pikey; Abraham Poulose; Peter Koulen Journal: Clin Ophthalmol Date: 2015-04-07