Precipitation of Nucleic Acids

Jones25 described the isolation of bacterial nucleic acids from Mycobacterium tuberculosis, My. phlei, and Sarcina lutea by precipitation with the 

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. 

With an extract of E. coli, the effectiveness in precipitating nucleic acids decreased in the order polylysine polyethyleneimine cetavlon streptomycin sulfate protamine sulfate MnCl2 spermine. With MnCl2, precipitation was relatively inefficient and as much as 75% nucleic acids remained in solution. Extracts of Micrococcus lysodiekticus treated with protamine sulfate showed a loss of catalase activity and ineffective removal of RNA. In contract, treatment of a similar extract with polyethyleneimine resulted in 90% precipitation of nucleic acids and 70% recovery of catalase 

A major drawback in use of polyethyleneimine is its suspected carcinogenic nature. Streptomycin sulfate is also toxic30 and its long-term use may result in development of resistant bacterial strains in the environment. 

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The high final concentrations of an analyte can result in at least two problems. First, precipitation of nucleic acids can be a problem at high concentrations. The experimentalist should be vigilant during sample preparation to ensure that no signs of precipitation are observed. Second, interactions between nucleic acids can still occur without precipitation. This possibility necessitates controls where BE-AES results are shown to be the same over a range of nucleic acid concentrations. 

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. 

This chapter will focus on a unique problem encountered during recovery of intracellulary produced proteins. Disruption of cells produces a mixture of nucleic acids and proteins in the solution from which the desired proteins must be fractionated. Typical separation schemes involve first the removal of nucleic acids from solution by precipitation. The desired protein is then isolated and purified from the mixture of remaining nucleic acids and proteins. A scheme for recovery of intracellular bacterial enzyme tartrate dehydrogenase from cell paste is shown in Fig. 1. Material balance at the different stages of the scheme in two different experiments showed that 53-60% loss in enzyme activity took place during precipitation of nucleic acids by protamine sulfate and during ammonium sulfate fractionation of proteins (Table 2). Reduction in volume, removal of major nonprotein... 

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. 

Conrad KM, Mast MG, BaU H et al. (1993) Concentration of liquid egg white by vacuum evaporation and reverse osmosis. J Food Sci 58(5) 1017-1020 Cordes RM, Sims WB, Glatz CE (1990) Precipitation of nucleic acids with poly(ethyleneimine). Biotechnol Prog 6 283-285... 

Cordes, R.M., Sima, W.B., Glatz, C.E. Precipitation of nucleic acids with poly (ethyleneimine). Biotechnol. Prog. 1990, 6 (4), 283-285. 

Atkinson, A., Jack, G.W. Precipitation of nucleic acids with polyethyleneimine and the chromatography of nucleic acids on immobilized polyethyleneimine. Biochem. Biophys. Acta 1973, 308 (1), 41-52. 

ATA forms a tight complex with RNA and DNA (ethanol precipitation of nucleic acids with ATA colors the pellet red), and apparently dissociates from the complex by electrophoresis. Excess ATA can be quickly removed by passage through a Sephadex G-50 spin column. ATA does not interfere with hybridizations of RNA samples nor does it affect spectrophotometry at 260 or 280 nm. However, ATA interferes with some restriction enzymes (e.g., HmdIII) and may also inhibit enzymes used in reverse transcription or in vitro translation. 

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. 

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. 

Nucleic acids may also be removed by treatment with nucleases, which catalyse the enzymatic degradation of these biomolecules. Indeed, nuclease treatment is quickly becoming the most popular method of nucleic acid removal during protein purification. This treatment is efficient, inexpensive and, unlike many of the chemical precipitants used, nuclease preparations themselves are innocuous and do not compromise the final protein product. 

Highly purified DNA may be obtained by repeating the chloroform-isoamyl alcohol extraction several times. The alcohol precipitation step may also be carried out many times. Various chromatographic methods including ion exchange have been applied to the purification of nucleic acids. 

Add 2 5x the volume of ethanol to precipitate labeled nucleic acids... 

Nucleic acids can be visualized by ethidium bromide staining, UV shadowing, or phosphorimaging of radioactive samples. A sterile scalpel should be used to excise the separated product which can then be eluted by electrophoresis (1 x Tris-borate, pH 8.3) into a 30kDa molecular weight cutoff (MWCO) filter (Millipore). The product is concentrated by centrifugation, dialyzed, and resuspended in the buffer of choice. The yield of nucleic acid is typically 75 %, and ethanol precipitation is not needed. 

The sensitivity of the detection is usually improved by the silver enhancement method. A better detection limit was reported when a silver enhancement method was employed, based on the precipitation of silver on AuNPs tags and its dissolution (in HNO3) and subsequent electrochemical potentiometric stripping detection [43]. The new silver-enhanced colloidal gold stripping detection strategy represented an attractive alternative to indirect optical affinity assays of nucleic acids and other biomolecules. 

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