Nucleic acids, separation ultracentrifugation

Absorption Optical System. During an ultracentrifuge experiment the solute, if denser than the solvent, sediments toward the bottom of the cell, thereby generating a concentration gradient. (There are many types of experiments the two most common types will be described later.) The instrument is constructed with two separate optical systems that record the concentration gradient during an experiment. One of these, the absorption optical system, is especially useful in biochemistry for the study of proteins and nucleic acids which have chromophores which absorb visible or uv light. 

Figure 1 represents a schematic illustration of the separation of ribosomes, sRNA, and the enzymes involved in the transfer reaction, described in detail below. At the end of the incubation period, the ribonucleoprotein and supernatant fractions were separated by ultracentrifugation, the perchloric acid-insoluble fraction was prepared from each, and the nucleic acids and proteins were isolated from the acid-insoluble residue (17). 

Identification and quantitative estimation of the components of nucleotide mixtures can be made by chromatographic techniques (which sometimes include the use of hydroxyapatite) (Figure 14.5). Nucleotide hydrolysis products can be separated by column chromatography and their amounts estimated by ultraviolet absorption, thus giving the overall base composition of the nucleic acid. Ultracentrifuging and gel electrophoresis based on polyacrylamide or agarose gels are also widely used. 

Isopycmc ultracentrifugation is one of the most commonly used procedures for separating nucleic acids. For DNA, the gradient is most often constructed with CsCl. The buoyant density, p, of double stranded Cs DNA depends on Its base composition ... 

The differences in buoyant density as well as molecular size and shape provide a means for separating the nucleic acids by isopycnic ultracentrifugation, a centrifugation technique in which particles sediment through a gradient medium (CsCl or sucrose) until they reach a zone of equal densities and band at that position. 

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