Purification, general nucleic acids

Affinity chromatography is similar in principle to the use of immuno-adsorbents for antibody and antigen purification (see Section IX, p. 375) and of insolubilized nucleic acids for purification of nucleic acids and related enzymes (see Section X, p. 384). The subject of affinity chromatography in general has been reviewed, ° and a mechanism... 

A prerequisite step to any rDNA work is the initial isolation of DNA or RNA from the source material (which can be microbial, plant, animal or viral). Numerous methodologies have been developed to achieve nucleic acid purification, and some of these methodologies have been adapted for use in a variety of commercially available purification kits. Although details vary, the general... 

ATP extraction has been used for the separation and large-scale purification of enzymes.45 47 Proteins and nucleic acids partition between the two phases differently due to differences in their surface charges and hydrophobic-hydrophilic domains. Proteins generally prefer the aqueous phase rich in... 

Purification of exopolysaccharides is generally a difficult matter, owing to the high viscosity of most polymers. Quaternary ammonium compounds, which precipitate acidic polysaccharides, have been used successfully247 to separate acidic from neutral polysaccharides. Pretreatment with enzymes that preferentially cleave such undesirable contaminants as protein, nucleic acid, and cells results in isolation of improved products. Selection either of polysaccharide or of contaminating material on either ion-exchange or affinity-chromatog-... 

Naturally occurring bacterial endotoxins contain the lipid, carbohydrate, and protein makeup of the outer cell membrane of GNB (Fig. 1). However, most of the commercial endotoxin preparations have been purified by various extraction procedures and are generally free of nucleic acids, proteins, phospholipids, and other bacterial cell components. The primary chemical configuration that remains after purification is apolysaccharide structure that is covalently bound to a lipid component called Lipid A. Based on its chemical nature, which is common to various bacterial families, this substance is referred to as lipopolysaccharide (LPS). Although the terms endotoxin and LPS are often used interchangeably, most reference endotoxin standards are purified preparations that are more correctly described as LPS. 

Nonviral formulations are generally more efficient and more toxic as the polymer-to-nucleic acid ratio is increased (higher N/P ratios), suggesting a role of free uncomplexed polymer in the solution in efficient delivery and toxicity. A recent report has shown that polyplexes of DNA and PEI contain an average of 3.5 plasmids (5800 base pairs) and 30 PEI (25 kDa) molecules when prepared at N/P ratios of 6 and 10, assuming that the DNA is completely complexed [267]. Based on these calculations, there is 86% of free PEI in the complex mixture [267,268]. Purification of the PEl/DNA complexes by dialysis has shown a reduction of toxcicity however, it also reduced transfection efficiency [269]. Efficient gene transfer was restored when free PEI was added to the mixture [269]. 

Ion-exchange chromatography is generally used in an early stage of biomacro-moleeular purification. It is used to separate neutral biomacromolecules (e.g. neutral polysaccharides) from charged biomaCTomolecules such as nucleic acids versus proteins. 

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