Fractionation methods nucleic acids

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... 

Treatment with hot organic solvents was the next step in the tissue fractionation, to remove lipid-phosphorous and breakdown lipid-protein interactions. In the Schneider procedure, nucleic acids were then extracted in hot dilute trichloroacetic or perchloric acid, leaving a protein residue with any phosphoprotein links still intact. This method was to become particularly useful when 3H thymidine became the preferred label for DNA in the early 1960s. For investigations where both RNA and DNA were to be examined the Schmidt-Thannhauser process was often chosen. Here the lipid-extracted material was hydrolyzed with dilute sodium hydroxide releasing RNA nucleotides and any hydroxyamino acid bound phosphorus. DNA could be precipitated from the extract but the presence in the alkaline hydrolysate of the highly labeled phosphate released from phosphoprotein complicated... 

Previous theoretical treatments of the transition between the helicel and random forms of the desoxyribose nucleic acid (DNA) molecule are extended to Include formally the explicit consideration of the dissociation into two separate chains and the consideration of the effects of the.ends of the chains, An approximate form for the fraction of the base pairs that are bonded is obtained in terms of two parameters, a stability constant for base pairing and a constant representing the interaction of adjacent base pairs. The matrix method of statistical mechanics proves to be adaptable to this problem. Some numerical examples are worked out for very long molecules, for which case it is found that the effect of concentration is small. 

The large UV absorptions of nucleic acids and proteins make the optical method a highly sensitive technique, and very small amounts of sample in very dilute solutions can be used. However, the selection of baselines between which the fractional change in absorption is evaluated must be made carefully to avoid error. The best agreement with values obtained from calorimetric measurements is observed when the slopes of both baselines are extended into the transition region, and the determination of x and y are made between the extrapolated lines as shown in Figure 16.6a. 

H. citelli, H. diminuta and H. microstoma, one of which is mitochondrial DNA (mtDNA) (394). The mtDNA of H. diminuta has been isolated (118) and has been shown to be a typical circular molecule. The characteristics of H. diminuta DNA are shown in Table 6.11. In contrast, E. multilocularis and E. granulosus produced two distinct DNA bands after fractionation in caesium chloride, but there was no evidence that the DNA from either band represented mtDNA (493). There is presumably so little mtDNA in comparison to nuclear DNA in these organisms that it is completely masked in preparations of total DNA by this method. That this is the case has been shown by a recent study (976), where a different procedure, based on the selective precipitation of nucleic acids by cetyltrimethylammonium bromide (CTAB), was employed to extract mtDNA from isolated mitochondria. Some 300 g and 50 g, respectively, of Taenia spp. and Echinococcus sp. tissue yielded approximately only 1 ng mtDNA. 

The earlier work on the isolation of PolyPs from the cells of living organisms usually employed the same methods as those used for the extraction of nucleic acids. It was not until 1936 that MacFarlane (MacFarlane, 1936) proposed a specific method for the extraction and fractionation of condensed phosphates present in cells. It was found that these phosphates could be divided into two main fractions, i.e. one soluble in 5 % trichloroacetic acid (TCA) and the other insoluble, and ever since then cellular condensed polyphosphates have been divided into acid-soluble and acid-insoluble fractions. 

To extract DNA and RNA from serum samples or from culture supernatant fractions which contain a low level of proviral DNA, the IsoQuick extraction kit (a modified guanidinium salt-organic solvent extraction method) by MicroProbe Corporation (Bothell, WA) has been used successfully by our group. Following the manufacturer s recommended procedures for total nucleic acid extraction, the final DNA-RNA suspension can be amplified directly to detect proviral DNA. Using cDNA templates synthesized from total nucleic acids with random hexamer priming generally increases the PCR product yield. 

The immunologically specific substance characteristic of Group C Meningococcus has already been mentioned this was extracted from phenol-killed organisms, and protein was removed by the Sevag method after fractionation with ethanol, nucleic acid was removed as the copper salt. The product contained 5 % of nitrogen and 11 % of acetyl, and hexosamine was the only constituent other than sialic acid. ... 

The solubility of proteins and nucleic acids in aqueous solution depends on the solvation of the macromolecule by water this can be influenced by pH, ionic strength and temperature, and also by the addition of salts, or water-soluble organic solvents. We discuss below the various precipitation methods that have been used with proteins and nucleic adds, particularly with regard to concentration and fractionation procedures. 

It is known that these polysaccharides display "remarkable surface adsorptive capacity both for organic and inorganic substances. It is therefore possible that these traces of nitrogen and phosphorus are due to strong adsorption between the polysaccharide and small amounts of protein or nucleic acid (or nucleic acid fragments). Such a system is resistant to the usual methods of fractionation, but a separation by electrophoretic means has been claimed. 

During inactivation steps, viral infectivity is reduced by treatment with chemicals and/or physical methods. Remnants of virus particles (e.g., viral nucleic acids) may remain in the product-containing fraction but are not infectious. Chemical methods of virus inactivation, such as treatment with solvent-detergent or acetone, must be placed upstream, since subsequent steps are needed to remove or reduce the levels of the toxic chemicals. Terminal inactivation is often achieved using physical methods, such as heat and low pH, because these methods leave no chemical residues. After treatment, the final products are delivered to patients, so aseptic processing conditions must be maintained throughout terminal inactivation steps and the parameters for virus inactivation must be balanced with the conditions to preserve product quality and yield. 

Chelex-100 (Whatman) resin was suspended in 1 M CuCl2 overnight, washed repeatedly in water and suspended in 1 N ammonia overnight. A column (0.9 x 45 cm) was packed with a small volume of non-CuCl2 treated resin at the bottom followed by the copper complexed resin above. After washing the column, the nucleic acid components were loaded in a small volume of water and eluted with water (nucleotides followed by weakly basic nucleosides), 1 N ammonia (other nucleosides) and/or 2.5 N ammonia (bases). The nucleotides are not bound by the column and so are not fractionated. The nucleosides and bases are, however, well fractionated. Several minor components are well separated. The method is relatively quick and the eluants are volatile. 

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