Rapid Method for Isolation of DNA from Glass Slide Smears for PCR

Monica Barbosa de Melo, Department of Clinical Medicine, State University of Campinas, CEP 13081, Campinas, SP (Brazil) Glass slide smears of cells from bone marrow aspirates or peripheral blood are widely used in pathology and he-matology laboratories for routine examination of patients. Since recent reports have shown the feasibility of DNA isolation from glass slide smears or tissues [1-3], we have devised a modification of these procedures for rapid DNA extraction from the glass slide smears of bone marrow or blood and its use for polymerase chain reaction (PCR). DNA was isolated from archived glass slide smears, unstained or stained with Wright’s stain. Typically, one or two glass slide smears were scraped into a sterile 1.5-ml Eppendorf tube using a new razor blade. The DNA was extracted from the resulting powder without phenol/chloroform extraction using a modification of a previously described method [4]: the scraped material was resuspended in 400 μl of 6 M guanidinium hydrochloride, 30 μl of 20% sodium sarcosyl, 30 μl of 7.5 M ammonium acetate and 10 μl of proteinase K (10 mg/ml). The mixture was heated at 60 °C for 1 h. If the powder was not completely dissolved, an additional 10 μl of proteinase Kwas added and the mixture was heated for another hour at 60 °C. The DNA was precipitated by addition of 1 ml of cold ethanol, gently homogenized and the mixture left at -20 °C overnight or 1 h at -70 °C. After centrifugation for 20 min the supernatant was discarded and the pellet resuspended in 50 μl of water. An aliquot of 1-5 μl was used for each amplification by polymerase chain reaction. The reaction mix consisted of 100 μl containing 100 pmol of each primer, 200 μM deoxy-nucleotides, 4 units of Taq Polymerase (BRL), 50 mMTris HC1 pH 8.3,0.01% gelatin, 1.5 mM HC1 and 1.5 mM MgCl2. The reaction mix was overlaid with light-mineral oil and PCR reactions were performed in a DNA thermal cycler (Perkin Elmer Cetus). The reaction conditions consisted