Nucleic Acid Solutions

This simple technique may also be applied to the sterilization of nonau-toclavable materials such as protein and nucleic acid solutions or heat-labile reagents. Bacterial contamination can be removed from these solutions by passing them through filter systems that have been sterilized by autoclaving. 

Microinjection involves the direct injection of nucleic acids into the nucleus or cytoplasm of target cells and is the simplest approach for gene delivery. Thin glass capillaries are used to inject nanoliters of nucleic acid solution into cells. One major drawback of this method is obviously the throughput every single cell has to be manipulated individually. This limits the use of microinjection to applications in which individual cell manipulation is possible, such as genetic engineering of... 

For very dilute nucleic acid solutions, precipitation with polyethylene glycol (PEG) is preferred. PEG 6000 is added to a concentration of 10% (w/v), and the solution is allowed to stand on ice for 2 h. The precipitate is removed by centrifugation and washed with 70% (v/v) ethanol. PEG can be used for fractional precipitation, since high molecular weight DNA is precipitated at lower PEG concentrations than low molecular weight DNA. It should be emphasised that oligonucleotides of chain length less than 20 cannot be precipitated effectively with either alcohol or PEG. 

Combine the diluted nucleic acid solution with the diluted cationic liposome prepared in separated tubes (in the example calculation, the total volume is 1,000 pi). Combine the dilutions in the prescribed order of protocol, since the order of dilution addition is important to achieve the optimal results (See Note 11). Mix by pipetting carefully up and down few times, or by gentle vortexing for 10 s to avoid precipitation, and let it stand for 10-45 min at room temperature to allow the nucleic acid-lipoplex formation. Depending on the concentration of nucleic acid and cationic liposome, the solution may appear cloudy. (See Note 12). 

Concentrate the nucleic acid solution by 2-butanol extraction. 

Reticulose. Reticulose is a peptide-nucleic acid solution in which ribonucleic acid is combined with short-chain peptides. Recently, reticulose has been described as an immune system modulator, and early clinical studies have shown that reticulose has an ability to rapidly inhibit the course of a variety of viral diseases, particularly in the acute stage. However, it has also been reported to be effective in sub-acute and chronic phases of certain infections and has exhibited no reported side effects in over 20 years of use. Retieulose appears to act by alteration of the host cell response to viral replication. It seems to stimulate the immune system via increases in endogenous interferon and increases in lymphocyte production. In clinical trials with patients infected with hepatitis A or B viruses, treatment with reticulose demonstrated positive results in that serum bilirubin levels reached normal levels and white blood eell count showed significant increases, indicating stimulation of the immune system (190). 

The availability of nucleic acid solutions that are uniform and free of precipitated material is crucial to the success of making mixtures having... 

As the temperature of a nucleic acid solution is raised, and the H bonds between the bases are broken, single strands are formed and there is an inerease in the ultra-violet absorption (hyperchro-mie effect) (Figure 10.46). For DNA this occurs over a eomparatively narrow range of temperature. 

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. 

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