3 - Working with nucleic acids

Summary

Before examining some of the specific techniques used in gene manipulation, it is useful to consider the basic methods required for handling, quantifying and analysing nucleic acid molecules. It is often difficult to make the link between theoretical and practical aspects of a subject, and an appreciation of the methods used in routine work with nucleic acids may be of help when the more detailed techniques of gene cloning and analysis are described.

Isolation of DNA and RNA

Every gene manipulation experiment requires a source of nucleic acid, in the form of either DNA or RNA. It is therefore important that reliable methods are available for isolating these components from cells. There are three basic requirements: (i) opening the cells in the sample to expose the nucleic acids for further processing,(ii) separation of the nucleic acids from other cell components, and (iii) recovery of the nucleic acid in purified form. A variety of techniques may be used, ranging from simple procedures with few steps, up to more complex purifications involving several different stages. These days, most biological supply companies sell kits that enable purification of nucleic acids from a range of sources.

The first step in any isolation protocol is disruption of the starting material, which may be viral, bacterial, plant or animal. The method used to open cells should be as gentle as possible, preferably utilising enzymatic degradation of cell wall material (if present) and detergent lysis of cell membranes.