Optimized Procedure for DNA Isolation from Fresh and Cryopreserved Clotted Human Blood Useful in Clinical Molecular Testing

Abstract

In the routine clinical laboratory, large amounts of uncoagulated blood are collected and the blood clot usually is discarded. In molecular biology, cells from EDTA-anticoagulated or acid-citrate-dextrose-anticoagulated peripheral blood are used as sources of DNA (1)(2)(3). After leukocyte isolation, most procedures utilize enzymatic cell digestion, followed by extraction with hazardous organic solvents (phenol-chloroform) and precipitation with ethanol (4)(5). To minimize the volume of blood collected for laboratory tests, several authors have developed methodologies to isolate DNA from blood clots (4)(5)(6)(7)(8). However, the techniques may be difficult or impractical and may require slicing of the clot with scalpels or other sharp instrument, exposing laboratory personnel directly to contaminated blood (4)(7). Other techniques are time-consuming, using many chaotropic reagents, enzymes, RNA-removal steps, or large volumes of samples and reagents not suitable in the clinical laboratory (4)(5)(6)(7)(8). We have optimized a nonenzymatic, nontoxic procedure for efficient DNA extraction from fresh and cryopreserved clotted blood. Blood samples were obtained from 24 unrelated individuals who had given informed consent. We compared 10 paired samples of EDTA-anticoagulated blood and fresh blood clot …