Extraction and Characterization of Bacterial DNA

The nucleic acids are among the most complex molecules that you will encounter in your biochemical studies. When the dynamic role that is played by DNA in the life of a cell is realized, the complexity is understandable. It is difficult to comprehend all the structural characteristics that are inherent in the DNA molecules, but most biochemistry students are familiar with the double-helix model of Watson and Crick. The discovery of the double-helical structure of DNA is one of the most significant breakthroughs in our understanding of the chemistry of life. This experiment will introduce you to the basic structural characteristics of the DNA molecule and to the forces that help establish the complementary interactions between the two polynucleotide strands. 

Now that these factors are understood, a general procedure of DNA extraction will be outlined. 

Step 1. Disruption of the cell wall and release of the DNA into a medium in which it is soluble and protectedfrom degradation. The isolation procedure de- 

Step 2. Dissociation of the protein-DNA complexes. Detergents are used 

Step 3. Separation of the DNA from other soluble cellular components. Before 

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For species characterization, whole-cell and extracellular proteins (CUnk and Pennington 1987 Berber et al. 2(X)3) or cellular fatty acid analysis and/or combined with gas chromatography may be applied. In general, fatty acid profiles correlate with the data on DNA homology and phenotype features (Behme et al. 1996). Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDl-TOF MS) was experimentally applied to intact bacterial cells or specimens prepared nsing extraction procedures. This chemotaxonomic method was used for identification of the staphylococcal species and MRSA strains (Du et al. 2(X)2 Carbonnelle et al. 2007). 

The distribution of manganese-dependent DNA synthesis and ribonucleotide reduction is currently being studied by Auling Because there is no simple assay for this type of reaction in bacterial extracts one has to follow the Mn dependence of DNA labeling in vivo and only in positive cases enzyme activity is then characterized in vitro. The five species of gram-positive bacteria identified in this way are included in Table 4. 

At the conclusion of the acclimation study, soils from the microcosms were used to inoculate enrichment media of die same composition as the treatments used in the acclimation study. The atrazine concentration was monitored by HPLC and the enrichments were subcultured when the atrazine concentration had declined by at least 25% relative to sterile controls. Enrichments were subcultured seven times, and then DNA was extracted from subsamples and subjected to PCR-DGGE analysis as in the acclimated soils. The DGGE profiles for soils and enrichment cultures difrmd. Sequencing results suggested that many phylotypes associated with afrazine treatments were not closely related to previously characterized atrazine-degrading bacterial isolates. 

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