Degradation and determination of nucleic acids

In order to study the nucleic acids by physicochemical or enzymological methods, they must be isolated and purified from other cell constituents. As their name indicates, they are acidic in character (one negative charge per nucleotide residue) and are neutralized by basic proteins (protamines, histones), polyamines (spermine, spermidine, etc.), or metallic cations (alkalis, earth alkalis). Nucleic acids are irreversibly denatured, if all their basic components are removed. For this reason, extractions are usually carried out in salt solutions buffered at pH 7. 

In a general procedure, after the cell wall is broken by mechanical or enzymatic methods (lysozyme), the resulting cell sap is treated with a protein-denaturing agent, such as phenol, or a detergent (dodecyl sulfate, lauryl sulfate), which precipitates proteins. Several extractions are frequently necessary. The final nucleic acid solution is treated with ethanol, to precipitate nucleic acids, or dialyzed against a suitable buffer solution. A review by Kirby (14) discusses the various isolation procedures used and their advantages and inconveniences. 

Hydrazinolysis yields apyrimidinic (i.e., DNA in which all the pyrimidines have been split at the glycosidic linkage). 

Dilute acid selectively cleaves the glycosidic linkage of purines in DNA, yielding apurinic acid. It does not act on RNA, except when hot and very concentrated (see Section 3.2.1). 

There exists a number of nucleases, some of which are highly specific (Fig. 3.8). One distinguishes between endonucleases, which attack within the nucleic acid chain, and exonucleases, which attack sequentially at a given end of the polynucleotide chain. 

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