Nucleic acids size-exclusion chromatography

Once the bacterial cells have been lysed and a crude preparation of plasmid has been obtained, several other methods are available for purification of plasmid from the crude nucleic acid preparation, such as cesium chloride ultracentrifugation, gel filtration/size exclusion chromatography, anion exchange (used in the Qiagen kit mentioned above), and HPLC (Table 2) (see Note 2). 

Names such as gel filtration chromatography (mobile phase is water), used by biochemists, and gel permeation chromatography (mobile phase is an organic solvent), used by polymer chemists, describe this technique. Size exclusion chromatography, however, is the recommended term. Molecular weight distribution of polymers can be obtained by this technique, and proteins, enzymes, peptides, nucleic acids, hormones, polysaccharides, and so on can be separated. 

Size Exclusion Chromatography of Nucleic Acids Yoshio Kato... 

In recent years, aqueous size exclusion chromatography applied In analytical studies of blomacromolecular systems has rapidly advanced In both theoretical and experimental aspects. The appearance of high performance gel chromatography materials has enabled us to separate proteins, nucleic acids and other blopolymer assemblies with high resolution. 

Dextran is an important extracellular bacterial polysaccharide widely used as a molecular sieve for purification and separation of biomacromolecules, like proteins, nucleic acids, and polysaccharides, as matrices (e.g., Sephadex ) in size-exclusion chromatography (Naessens et al. 2005). Dextran is also used in clinical research as clinical dextran, a blood plasma substitute, in alleviate iron-deficiency anemia, and in confectionary to improve moisture retention, viscosity, and inhibit sugar crystallization (Sutherland 1997,1998,1999 Kumar et al. 2007 Kumar and Mody 2009). 

In research conducted by Sabbioni and Girardi, NAA was developed for the simultaneous analysis of heavy metals in microsamples of potential metal binding components such as metalloenzymes (calf intestine alkaline phosphatase, cow milk xanthine oxidase, calf thymus deoxynucleotidyltransferase) and nucleic acid (calf thymus DNA), purified by size-exclusion chromatography and ion-exchange chromatography. 

In ion-exchange chromatography nucleic acids are separated on the basis of the difference in charge. Each nucleic acid has a different net charge based on the number of phosphate groups in the molecule (base length) and the respective charges on the bases (base composition). The latter effect is relatively minor and so separation depends almost exclusively on size. Separation... 

E. Sephadex Sephadex layers are prepared from modified dextran gels for the separation of hydrophilic solutes such as nucleic acids and peptides. The mechanism of separation is partition chromatography governed by size exclusion in the swollen gel containing pores of controlled dimensions. The gels, a layer spreader and special equipment for developing layers are available from Pharmacia Fine Chemicals. 

The predominant feature of nucleic acids in aqueous solution is the ionic nature of their phosphate groups which makes these molecules ideal candidates for separation by ion-exchange chromatography. Reversed phase separations may also be carried out by utilising the weak hydrophobic nature of the nucleic acid bases. Natural nucleic acids occur in many different size classes and group separations (e.g. between ribosomal RNA and transfer RNA) by size exclusion can also be effective. The relative merits of these methods are described in the following sections. 

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