Recovery of DNA from soils and sediments.
Experiments were performed to evaluate the effectiveness of two different methodological approaches for recovering DNA from soil and sediment bacterial communities: cell extraction followed by lysis and DNA recovery (cell extraction method) versus direct cell lysis and alkaline extraction to recover DNA (direct lysis method). Efficiency of DNA recovery by each method was determined by spectrophotometric absorbance and using a tritiated thymidine tracer. With both procedures, the use of polyvinylpolypyrrolidone was important for the removal of humic compounds to improve the purity of the recovered DNA; without extensive purification, various restriction enzymes failed to cut added target DNA. Milligram quantities of high-purity DNA were recovered from 100-g samples of both soils and sediments by the direct lysis method, which was a greater than 1-order-of-magnitude-higher yield than by the cell extraction method. The ratio of labeled thymidine to total DNA, however, was higher in the DNA recovered by the cell extraction method. than by the direct lysis method, suggesting that the DNA recovered by the cell extraction method came primarily from active bacterial cells, whereas that recovered by the direct lysis method may have contained DNA from other sources.
Steffan
R J
RJ
Department of Biology, University of Louisville, Kentucky 40292.
Goksøyr
J
J
Bej
A K
AK
Atlas
R M
RM
D002469:Cell Separation; D004269:DNA, Bacterial / Q000302:isolation & purification*; D017886:Durapatite; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D015151:Immunoblotting; D008722:Methods; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology
D004269:DNA, Bacterial; D006882:Hydroxyapatites; D012987:Soil; D017886:Durapatite