In the previous two chapters I examined the two essential components of genetic engineering, these being (i) the ability to cut, modify and join DNA molecules in vitro, and (ii) the host/vector systems that allow recombinant DNA molecules to be propagated. With these components at his or her disposal, the genetic engineer has to devise a cloning strategy that will enable efficient use of the technology to achieve the aims of the experiment. In Chapter 1 I showed that there are basically four stages to any cloning experiment (Fig. 1.1), in volving generation of DNA fragments, joining to a vector, propagation in a host cell, and selection of the required sequence. In this chapter I examine some of the strategies that are available for completing the first three of these stages by the traditional methods ogfene cloning, largely restricting the discussion to cloning eukaryotic DNA in E. coli. The use of the polymerase chain reaction (PCR) in amplification and cloning of sequences is discussed in Chapter 7, as this is now a widely used protocol which in some cases bypasses standard cloning techniques. Selection of cloned sequences is discussed in Chapter 8, although the type of selection method that will be used does have to be considered when choosing host/vector combinations for a particular cloning exercise.

Which approach is best?

The complexity of any cloning experiment depends largely on two factors: (i) the overall aims of the work, and (ii) the type of source material from which the nucleic acids will be isolated for cloning.