Nucleic acids isolation from bacteria

An integrated pTAS system for the detection of bacteria including lysis, DNA purification, PCR and fluorescence readout has also been published recently [113]. A microfluidic plastic chip with integrated porous pol5mier monoliths and silica particles for lysis and nucleic acid isolation was used for detection (Fig. 8). A custom-made base device provided liquid actuation and off-chip valving by stopping liquid flow from the exits of the chip, utilizing the incompressibility of liquids. Detection of 1.25 x 10 cells of B. subtilis was demonstrated with all assay steps performed on-chip. 

Of all the various nucleic acid molecules within the cell the three that are most often isolated are chromosomal DNA, mRNA from tissue or cells and plasmid DNA from bacteria. In all cases the principles for isolation are similar and can be divided into three stages. 

Membrane teichoic acids have now been detected in a large number of bacteria, including almost all of the Gram-positive organisms examined. Nevertheless, the proportion present is sometimes small, and separation of the acids from other macromolecular cell-components, such as nucleic acids, peptides, and polysaccharides, is difficult. Consequently, few have been obtained in an amount and of a purity adequate for detailed chemical study. Even from the limited studies so far made, it is clear that structural details differ considerably from case to case, and it is convenient to classify these teichoic acids according to the organisms from which they have been isolated. 

Another strategy exploits the nucleic acids from diverse organisms where it is not necessary to isolate the bacteria themselves. This approach has particular benefits because only a small fraction of organisms in a particular habitat is culturable (Table 3.4). 

A number of studies on photochemistry of the nucleic acid bases in aqueous solutions demonstrated that while uracil undergoes reversible hydration under exposure to UV irradiation, the other bases (thymine, adenine, and guanine) were stable [41,42], However, the sensitivity of dissolved thymine to UV irradiation can be significantly increased if the solution is rapidly frozen [43]. In 1960 the thymine photoproduct was isolated from irradiated frozen aqueous solution of thymine. Elemental analysis, molecular weight measurements, powder X-ray diffraction, NMR and IR spectroscopy confirmed that the most likely photoproduct is a thymine dimer [20]. Similar photoproduct was obtained by hydrolysis of irradiated DNA. Its formation was attributed to reaction between two adjacent thymine groups on the same DNA chain [44], Independently an identical compound was isolated from DNA of UV-irradiated bacteria [45]. 

Sources from which desoxyribosenucleic acid has been isolated include fish sperm, thymus, spleen, pancreas, testicles, placenta, mammary glands, brain, liver, kidney, blood cells, thyroid, intestines, lungs, lymphatic glands, bacteria, and tumor tissue. It is difficult to obtain a uniform product, free from protein the first serviceable method was that of Neumann, which has been improved upon by later workers. The nucleic acid from thym,us glands may be isolated in the following manner ... 

Several kits are commercially available for the extraction of RNA from plant, bacteria, or mammalian sources. We have used the RNeasy Mini Kit (Qiagen, Valencia, CA) for the extraction of RNA from bacteria following the procedure recommended by the manufacturer (92). This kit relies on guanidine thiocyanate-silica isolation of RNA. Here, guanidine thiocyanate serves as a chaotropic agent, which both lyses cells and inactivates nucleases. In its presence, the released nucleic acids bind to silica particles, which provide a solid phase from which the collected RNA can later be eluted using water (93). This kit is suitable for the isolation and purification of up to 100 pg RNA molecules >200... 

Less than 3% of soil organic phosphorus is present as nucleic acids and derivatives derived from the decomposition of living organisms (Dalai, 1977). The four bases of DNA have been identified in humic acids (Anderson, 1961). The presence of nucleic acids and derivatives in the soil was confirmed by the isolation of two pyrimidine nucleoside diphosphates (Anderson, 1970). Nucleic acids are rapidly mineralized, re-synthesized and combined with other soil constituents, or incorporated into microbial biomass (Anderson and Malcolm, 1974). Nevertheless, the interaction of nucleases with soil constituents can inhibit DNA hydrolysis, with important environmental consequences related to extracellular gene uptake by bacteria (Demanfeche et al., 2001). 

Jones, A. S. and Walker, R. T. (1968) The isolation of nucleic acids from grampositive bacteria. Arch. Biochem. Biophys. 128, 579-582. 

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