More on cloning
The difficulty of the task of isolating the desired recombinant from a population of transformed bacteria depends on the cloning strategy one adopts.
Presently, there are three possible cloning methods;
each has its own advantages and disadvantages. These are shotgun
cloning, copy DNA (cDNA) cloning and direct gene sythesis.
As to the methods of screening these recombinants, one could employ genetic methods, immunochemical methods and methods based on nucleic acid hybridization.
More often, the objective of cloning foreign proteins by recombinant DNA
is a commercial product; it is then essential to
maximize gene expression. This can be done by the following ways:
increase the number of copies of the plasmid vector per unit cell
use a stronger promoter to be able to synthesize more mRNA molecules
use the optimum sequnce of rbs and flanking DNA; a single base change
can affect the translation by 1000-fold
proper choice of codon in the cloning gene
using highly stable recombinants which do not mutate to avoid loss
of recombined DNA plasmids.
sythesize the proteins with the the appropriate protection from the reagents used
Today, recombinant DNA technology has been applied extensively in the
large scale production of proteins and enzymes that are of therapeutic
and diagnostic interest. The most applied expression system is that of
E. coli but a lot of research looks at animal and mamalian cell
cultures because of certain limitations encountered in bacterial systems.
Examples of important products of recombinant DNA technology are:
human insulin
human growth hormone
tissue plasminogen activator
factor viii
protein c
protein a
erythropoeitin
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