Nucleic acids precipitants

Higgins et al.35 used manganous sulfate for precipitation of nucleic acids in continuous production of /3-galactosidase from Escherichia coli. The precipitation was fast (most of the nucleic acid was precipitated in 10 min of contact time). At 0.05 M manganous sulfate, only a small amount of nucleic acid precipitated. Precipitation was improved at 0.1 and 0.5 M concentrations of the precipitant, but as much as 70% enzyme activity was also lost under these conditions. 

FIGURE 2 Precipitation of nucleic acids with streptomycin sulfate (A) effect on nucleic acid precipitation and residual TDH activity in the supernate and (B) effect on the ratio of TDH lost to nuclei acid precipitated. 

Also, as a word of caution, it should be noted that nucleic acids precipitated by PEG can contain macromolecular impurities originating from the PEG these impurities are not detectable by the usual methods (e.g., electrophoresis or UV absorption spectroscopy) and they cannot be readily removed even by CsCl gradient centrifugation. 

Derivation Hydrolysis of nucleic acids, precipitation from urea and ethyl formylacetate. Radioactive forms available. 

These differences suggest several advantages of the chemical permeabilization method. First, avoiding cell breakage should simplify the cell removal step. Second, retention of the nucleic acids inside the cell should eliminate the need for a nucleic acid precipitation step. Another advantage is that the permeabilization process also kills the cells thereby eliminating the need for the federally mandated cell killing step. 

Purification of b-galactosidase from E. coli may be compared Higgins (26) outlines a process of succeeding centrifugations to remove cell debris, nucleic acids precipitate, and protein precipitate (product). Veide (23) outlines a single aqueous extraction with PEG and salt in which b-galactosidase partitions to the PEG-rich phase. Cells, nucleic acids, and a major part of the contaminating proteins partition to the salt-rich phase. 

Extraction of proteia requires breaking the cell wall to release the cytoplasmic contents. This can be achieved by high speed ball or coUoid mills or by high pressure (50—60 Mpa) extmsion. Proteia is extracted by alkaline treatment followed by precipitation after enzymatic hydrolysis of nucleic acids. Although the proteia can be spun iato fibers or texturized, such products are more expensive than those derived from soybean and there is no market for them. 

The presence of nucleic acids ia yeast is oae of the maia problems with their use ia human foods. Other animals metabolize uric acid to aHantoia, which is excreted ia the uriae. Purines iagested by humans and some other primates are metabolized to uric acid, which may precipitate out ia tissue to cause gout (37). The daily human diet should contain no more than about 2 g of nucleic acid, which limits yeast iatake to a maximum of 20 g. Thus, the use of higher concentrations of yeast proteia ia human food requires removal of the nucleic acids. Unfortunately, yields of proteia from extracts treated as described are low, and the cost of the proteia may more than double. 

On homogenization, the lysate may drastically increase in viscosity due to DNA release. This can be ameliorated to some extent using multiple passes to reduce the viscosity. Alternatively, precipitants or nucleic acid digesting enzymes can be used to remove these viscosity-enhancing contaminants. 

According to Barbieri et al., it can not be excluded that some of the coordination sites are occupied by (Nj atoms of the nucleic acid constituents. The precipitate obtained from [Ph2Sn(IV)] would contain both the [R2Sn(IV)] -DNA complex and distannoxane [(Ph2SnCl)20]. The main products of the... 

Classical gene transfer methods still in use today are diethylamino ethyl (DEAE)-dextran and calcium phosphate precipitation, electroporation, and microinjection. Introduced in 1965, DEAE-dextran transfection is one of the oldest gene transfer techniques [2]. It is based on the interaction of positive charges on the DEAE-dextran molecule with the negatively charged backbone of nucleic acids. The DNA-DEAE-dextran complexes appear to adsorb onto cell surfaces and be taken up by endocytosis. 

Patients with tumor lysis syndrome experience a wide range of metabolic abnormalities. The massive cell lysis that occurs leads to the release of intracellular electrolytes, resulting in hyperkalemia and hyperphosphatemia. High concentrations of phosphate bind to calcium, leading to hypocalcemia and calcium phosphate precipitation in the renal tubule. Purine nucleic acids are also released that are subsequently metabolized to uric acid... 

A technical challenge with this step is to achieve RNA extraction of uniform quality and efficiency for each fraction. This is because the amount of RNA in each sucrose gradient fraction varies considerably and the high concentration of sucrose in the bottom fractions interferes with phase separation in typical phenol-based extraction steps. To address these problems, we spike each fraction with an aliquot of a foreign (control) RNA, which can be used later to correct for differences in RNA recovery (and reverse transcription efficiency) between samples. We then remove sucrose from the samples by precipitation of total nucleic acid and protein with ethanol. To purify RNA, a standard Trizol (Invitrogen) extraction is performed as outlined later (also see product insert). 

Four processes are concerned in the isolation of a nucleic acid. First is the destruction of the tissue structure (stage 1). A nucleoprotein complex is then separated from other cellular constituents (stage 2). This complex is dissociated and the protein is removed (stage 3) and, finally, the nucleic acid is precipitated from the resulting solution (stage 4). Disintegration of... 

In a number of methods, isolation of the nucleoprotein complex (stage 2) is avoided. In the isolation of ribonucleic acid from beef pancreas,1241 nuclear material and cell debris are removed from a normal-saline extract of the minced tissue, which is then brought to half-saturation with sodium chloride (to dissociate the protein from the nucleic acid). After removal of the protein, the nucleic acid is precipitated with alcohol. However, the suggestion has been made126 that it is more satisfactory to isolate the nucleoprotein first, and this has been carried out, for instance, in the extraction of the ribonucleic acid from fowl sarcoma GRCH 15.126 Nucleoprotein complexes have also been isolated from baker s yeast127 and have been separated into various fractions, the nucleic acids from which differ slightly in composition. In addition, nucleoproteins have been isolated by complex formation with cetyltrimethylammonium bromide.128... 

Pillai and Ganguly [286] have concentrated the nucleic acids from seawater by adsorption on homogeneously precipitated barium sulfate, then hydrolysed with 0.02 M hydrochloric acid and analysed for the constituents. 

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