Nucleic acids digestion

On homogenization, the lysate may drastically increase in viscosity due to DNA release. This can be ameliorated to some extent using multiple passes to reduce the viscosity. Alternatively, precipitants or nucleic acid digesting enzymes can be used to remove these viscosity-enhancing contaminants. 

Analytical Techniques and Physical Methods.— The mapping of oligonucleotides and nucleic acid digests on cellulose or cellulose-polyethyleneimine has been described recently, and columns of mercurated dextran or dihydroxyborylcellulose have been used to fractionate nucleotide mixtures. Electrophoresis on polyacrylamide gels has been advocated as a rapid method for desalting and fractionating mixtures of oligonucleotides. ... 

Especially in the fields of amino acids and peptides, such as they are obtained by enzymatic hydrolysis of proteins, a two-dimensional combination of paper or thin-layer chromatography and electrophoresis has become very popular. The patterns of tryptic hydrolysates are termed hnger-prints and can reveal abnormal peptides if mutated anomalous proteins are compared with their normal counterparts. Similar finger-prints can be obtained from nucleic acid digests. 

The removal and reduction of the nucleic acid content of various SCPs is achieved by chemical treatment with sodium hydroxide solution or high salt solution (10%). As a result, crystals of sodium urate form and are removed from the SCP solution.16,17 The quality of SCP can be upgraded by the destruction of cell walls. That may enhance the digestibility of SCP. With chemical treatment the nucleic acid content of SCP is reduced. 

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

Protoplasmic structures are not very stable. Consequently, if protective agents were not employed, destruction of the walls was accompanied by a rapid lysis of the protoplasts, followed by the liberation of most of the cell protein and nucleic acid in soluble form. This could be prevented by employment of the enzyme in a 0.2 M solution of sucrose or cane sugar. After digestion of the cell walls, the living protoplasts rounded up into spheres. 

The tissue or cell sample is firstly homogenized in a buffer containing a detergent such as Triton X-100 and sodium deodecyl sulphate (SDS), which disrupts the cell and dissociates DNA-protein complexes. Protein and RNA are then removed by sequential incubations with a proteolytic enzyme (usually proteinase K) and ribonuclease. Finally the DNA is extracted into ethanol. Ethanol only precipitates long chain nucleic acids and so leaves the single nucleotides from RNA digestion in the aqueous layer. 

Digestion of endogenous nucleic acids (cell death, RNA turnover)... 

Hyperuricemia may be produced by overproduction of uric acid or under-excretion of uric add by the kidneys. Kyperuricemia may progress to acute and chronic gouty arthritis if uric acid (monosodium urate) is deposited in joints and surrounding soft tissue, where it causes inflammation, Uric add is produced from excess endogenous purines as shown in Figure 1-18-5, and is also produced from dietary purines (digestion of nucleic acid in the intestine) by intestinal epithe-lia. Both sources of uric acid are transported in the blood to the kidneys for excretion in urine. 

The nutrients discussed in this section are carbohydrate, fat, protein and nucleic acids. The nucleic acids, upon digestion, provide phosphate, bases and nucleosides (Chapters 10 and 20). Each is discussed under four separate headings ... 

In common with the digestion of other macromolecules, nucleic acids are hydrolysed in a stepwise manner, by pancreatic nuclease (diesterase enzymes) which hydrolyse the bonds between two adjacent phosphate groups in RNA and DNA. The resultant oligoribonucleotides and oligode-oxy ribonucleotides are hydrolysed to form nucleoside monophosphates, which lose their phosphate to form nucleosides, by the action of pancreatic phosphatase. In brief, the process is ... 

Cell debris may be removed by centrifugation at 10,000 g for 30 min. The nucleic acids being the major contaminant can be removed by precipitation with a positively-charged pol)uner such as polyethyleneimine PEI (t)q)ically 0.5-1% of a 10% solution). Addition of magnesium to the resuspension buffer will assist in the enzyme digestion of DNA by DNAse. Some loss of protein may occur by copreciptation, which is especially the case with some DNA-binding proteins. This can usually be avoided by a 1 1 dilution of the crude extract with buffer. 

---