Principles of Gene Manipulation

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Contents and supplementary information for: Principles of Gene Manipulation Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 Gene manipulation: an all-embracing technique Basic techniques - (POGC02.pdf, 1,560KB) Cutting and joining DNA molecules Basic biology of plasmid and phage vectors Cosmids, phasmids and other advanced vectors Cloning strategies Additional updated information on Cloning strategies Sequencing and mutagenesis Cloning in bacteria other than E. coli Cloning in Saccharomyces cerevisiae and other fungi Gene transfer to animal cells Additional updated information on Gene transfer to animal cells Chapter 11 Genetic manipulation of animals Additional updated information on Genetic manipulation of animals Chapter 12 Chapter 13 Gene transfer to plants Additional updated information on Gene transfer to plants Advances in transgenic technology Additional updated information on Advances in transgenic technology Chapter 14 or (POGC13.pdf - size: 353KB)

Applications of recombinant DNA tecnology

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CH A P T E R 1

Gene manipulation: an all-embracing technique
Introduction
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.

Sequence analysis
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...
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