|SREL Reprint #1926|
Comparison of Methods of DNA Extraction from Stream Sediments
LAURA G. LEFF,2 JAMES R. DANA,1 J. VAUN McARTHUR,2 AND LAWRENCE J. SHIMKETS2
Department of Microbiology, University of Georgia, Athens, Georgia 306021 and Savannah River Ecology Laboratory, Aiken, South Carolina 298022
In Upper Three Runs Creek (Aiken, S.C.) and many other environments, less
than 1% of bacteria visible microscopically can be cultured. Exploitation 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 study was to compare three published
methods of DNA extraction that fall into two general categories: those in which
cells are lysed in sediments (the Ogram and Tsai and methods [A. Ogram, G. S.
Sayler, and T. Barkay, J. Microbiol. Methods 7:57-66, 1987; Y. L. Tsai and B.
H. Olson, Appl. Environ. Microbiol. 57:1070-1074, 19911) and those in which
cells are removed from sediments prior to lysis (the Jacobsen method [C. S.
Jacobsen and 0. S. Rasmussen; Appi. Environ. Microbiol. 58:2458-2462,
1992]). DNA yield varied with extraction method; the Ogram method had a
significantly higher yield than the other methods. However, DNA extracted via
the Ogram method was badly sheared and contained a smaller proportion of
eubacterial DNA. The Tsai method was less time consuming 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 recommended because of the low
concentration of contaminants. If DNA is to be used directly in DNA-DNA
hybridizations, the Ogram method is recommended since it gives maximal yields.
However, if a Southern blot is to be performed, the Tsai method is
recommended because of the high degree of DNA fragmentation observed with
the other methods.
SREL Reprint #1926
Leff, L.G., J.R. Dana, J.V. McArthur, and L.J. Shimkets. 1995. Comparison of methods of DNA extraction from stream sediments. Applied and Environmental Microbiology 61:1141-1143.