4 - The tools of the trade

Summary

The genetic engineer needs to be able to cut and join DNA from different sources. In addition, cer tain modifications may have to be carried out to the DNA during the various steps required to produce, clone and identify recombinant DNA molecules. The tools that enable these manipulations to be performed are enzymes, which are purified from a wide range of organisms and can be bought from various suppliers. In this chapter I examine some of the important classes of enzymes that make up the genetic engineer's toolkit.

Restriction enzymes – cutting DNA

The restriction enzymes, which cut DNA at fidened sites, represent one of the most important groups of enzymes for the manipulation of DNA. These enzymes are found in bacterial cells, where they function as part of a protective mechanism called the restriction–modification system. In this system the restriction enzyme hydrolyses any exogenous DNA that appears in the cell. To prevent the enzyme acting on the host cell DNA, the modification enzyme of the system (a methylase) modifies the host DNA by methylation of particular bases in the recognition sequence, which prevents the restriction enzyme from cutting the DNA.

Restriction enzymes are of three types (I, II or III). Most of the enzymes used today are type II enzymes, which have the simplest mode of action. These enzymes are nucleases (see Section 4.2.1), and as they cut at an internal position in a DNA strand (as opposed to beginning degradation at one end) they are known as endonucleases.