Simple, rapid method for direct isolation of nucleic acids from aquatic environments.
Direct isolation of nucleic acids from the environment may be useful in several respects, including the estimation of total biomass, detection of specific organisms and genes, estimations of species diversity, and cloning applications. We have developed a method that facilitates the concentration of microorganisms from aquatic samples and the extraction of their nucleic acids. Natural water samples of 350 to greater than 1,000 ml are concentrated on a single cylindrical filter membrane (type SVGS01015; Millipore Corp., Bedford, Mass.), and cell lysis and proteolysis are carried out within the filter housing. Crude, high-molecular-weight nucleic acid solutions are then drawn off the filter. These solutions can be immediately analyzed, concentrated, or purified, depending on the intended application. The method is simple, rapid, and economical and provides high-molecular-weight chromosomal DNA, plasmid DNA, and speciated RNAs which comigrate with 5S, 16S, and 23S rRNAs. The methods presented here should prove useful in studying both the ecology and the phylogeny of microbes that resist classical culture methods.
Somerville
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Center of Marine Biotechnology, University of Maryland, Baltimore 21202.
Knight
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Straube
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Colwell
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D000085:Acetates; D000333:Aeromonas / Q000235:genetics; D001419:Bacteria / Q000235:genetics*; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D000431:Ethanol; D010957:Plasmids; D012329:RNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D015183:Restriction Mapping; D014733:Vibrio / Q000235:genetics; D014734:Vibrio cholerae / Q000235:genetics; D014737:Vibrionaceae / Q000235:genetics; D014871:Water Microbiology
D000085:Acetates; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D000431:Ethanol; C018824:ammonium acetate