COMPARISON OF METHODS OF DNA EXTRACTION FROM STREAM SEDIMENTS

In Upper Three Runs Creek (Aiken, S.C.) and many other environments, l ess than 1% of bacteria visible microscopically can be cultured. Explo itation of molecular biology techniques has led to development of new methods, such as extraction of nucleic acids from soils or sediments, to study the dominant, nonculturable bacteria. The purpose of this stu dy was to compare three published methods of DNA extraction that fall into two general categories: those in which cells are lysed in sedimen ts (the Ogram and Tsai and methods [A. Ogram, G. S. Sayler, and T. Bar kay, J. Microbiol. Methods 7:57-66, 1987; Y. L. Tsai and B. H. Olson, Appl. Environ. Microbiol. 57:1070-1074, 1991]) and those in which cell s are removed from sediments prior to lysis (the Jacobsen method [C. S . Jacobsen and O. S. Rasmussen; Appl. Environ. Microbiol. 58:2458-2462 , 1992]). DNA yield varied with extraction method; the Ogram method ha d a significantly higher yield than the other methods. However, DNA ex tracted via the Ogram method was badly sheared and contained a smaller proportion of eubacterial DNA. The Tsai method was less time consumin g than the other methods, but DNA samples were of lower purity. If DNA purity is of paramount concern (as would be the case if PCR was to be performed) and quantity is not important, the Jacobsen method is reco mmended because of the low concentration of contaminants. If DNA is to be used directly in DNA-DNA hybridizations, the Ogram method is recom mended since it gives maximal yields. However, if a Southern blot is t o be performed, the Tsai method is recommended because of the high deg ree of DNA fragmentation observed with the other methods.