Comparison of methods of DNA extraction from stream sediments.
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, 1991]) and those in which cells 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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Leff
L G
LG
Department of Microbiology, University of Georgia, Athens 30602.
Dana
J R
JR
McArthur
J V
JV
Shimkets
L J
LJ
D001419:Bacteria / Q000302:isolation & purification; D001433:Bacteriolysis; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D008969:Molecular Sequence Data; D012988:Soil Microbiology; D014867:Water / Q000737:chemistry
D004269:DNA, Bacterial; D014867:Water