Direct extraction of DNA from soils for studies in microbial ecology.
Molecular analyses for the study of soil microbial communities often depend on the extraction of DNA directly from soils. These extractions are by no means trivial, being complicated by humic substances that are inhibitory to PCR and restriction enzymes or being too highly colored for blot hybridization protocols. Many different published protocols exist, but none have been found to be suitable enough to be generally accepted as a standard. Most direct extraction protocols start with relatively harsh cell breakage steps such as bead-beating and freeze-thaw cycles, followed by the addition of detergents and high salt buffers and/or enzymic digestion with lysozyme and proteases. After typical organic extraction and alcohol precipitation, further purification is usually needed to remove inhibitory substances from the extract. The purification steps include size-exclusion chromatography, ion-exchange chromatography, silica gel spin columns, and cesium chloride gradients, among others. A direct DNA extraction protocol is described that has been shown to be effective in a wide variety of soil types. This protocol is experimentally compared to several published protocols.
Schneegurt
Mark A
MA
Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA mark.schneegurt@wichita.edu
Dore
Sophia Y
SY
Kulpa
Charles F
CF
D004247:DNA / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000032:analysis / Q000302:isolation & purification; D004463:Ecology; D004586:Electrophoresis; D005821:Genetic Techniques; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology
D004275:DNA, Ribosomal; D012987:Soil; D004247:DNA