Applications of recombinant DNA tecnology
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CH A P T E R 1
Gene manipulation: an all-embracing technique
Occasionally technical developments in science occur that enable leaps forward in our knowledge and increase the potential for innovation. Molecular biology and biomedical research experienced such a revolutionary change in the mid-70s with the development of gene manipulation. Although the initial experiments generated much excitement, it is unlikely that any of the early workers in the ﬁeld could have predicted the breadth of applications to which the technique has been put. Nor could they have envisaged that the methods they developed would spawn an entire industry comprising several hundred companies, of varying sizes, in the USA alone. The term gene manipulation can be applied to a variety of sophisticated in vivo genetics as well as to in vitro techniques. In fact, in most Western countries there is a precise legal deﬁnition of gene manipulation as a result of government legislation to control it. In the UK, gene manipulation is deﬁned as the formation of new combinations of heritable material by the insertion of nucleic acid molecules, produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation. The deﬁnitions adopted by other countries are similar and all adequately describe the subject-matter of this book. Simply put, gene manipulation permits stretches of DNA to be isolated from their host organism and propagated in the same or a different host, a technique known as cloning. The ability to clone DNA has far-reaching consequences, as will be shown below.
Cloning permits the isolation of discrete pieces of a genome and their ampliﬁcation. This in turn enables the DNA to be sequenced. Analysis of the sequences of some genetically well-characterized genes led to the identiﬁcation of the sequences and structures which characterize the principal control elements of gene expression, e.g. promoters, ribosome binding sites, etc. As this information built up it became possible to scan new DNA sequences and identify potential new genes, or open reading frames, because they were bounded by characteristic motifs. Initially this sequence analysis was done manually but to the eye long runs of nucleotides have little meaning and patterns evade recognition. Fortunately such analyses have been facilitated by rapid increases in the power of computers and improvements in software which have taken place contemporaneously with advances in gene cloning. Now sequences can be scanned quickly for a whole series of structural features, e.g. restriction enzyme...