Gene Cloning

58 Gene Cloning
fulfill many specialized functions. Two of the features of pUC18, the multiple
cloning site and the facility to use blue–white selection, have been
included in many different vectors. There are many different multiple
cloning sites offering a wide range of unique restriction sites for cloning
into and these are incorporated into a wide range of vectors. The α peptide
of the lacZ gene is also incorporated into many vectors, either for blue–
white selection for DNA inserts as in the pUC vectors, or as a reporter gene
for monitoring gene expression (Chapter 10).
In constructing DNA libraries from eukaryotic organisms with large
genomes (Chapter 4) it is often necessary to be able to clone very large DNA
fragments. Standard plasmid cloning vectors tend to become unstable with
very large inserts; however, plasmids have been constructed, based on the
F plasmid origin of replication which can accommodate up to 300 kb of
insert DNA, these so called bacterial artificial chromosomes (BACs) are discussed
in detail in Section 4.16.
Many applications of gene cloning involve cloning a gene in order to
express the gene and to produce the protein which it encodes. This is true
of many biotechnological and medical applications of gene cloning where
commercially useful or therapeutic proteins are produced from cloned
genes (Chapters 9 and 13) but is equally true in a research context where
gene cloning is an important tool in the functional analysis of proteins
(Chapter 10). A whole range of cloning vectors are available where precise
control of expression of the cloned gene is possible so that there is no background
expression, but when the gene is induced gene expression is rapid
and large quantities of protein are produced. These are discussed in detail
in Section 9.3.
3.10 Analyzing Cloned DNA by Restriction Mapping
Plasmid vectors that allow you to identify recombinant clones by looking
for insertional inactivation of either antibiotic resistance genes or lacZ are
very useful. However, potential recombinant clones still need to be analyzed
by restriction mapping. This means purifying the plasmid DNA from
individual clones, cutting it with restriction enzymes, and analyzing the
sizes of the fragments produced. To take a simple example, if you have
cloned a 1 kb EcoRI fragment of genomic DNA into the EcoRI site in the
multiple cloning site of pUC18 you would expect clones to be resistant to
ampicillin and lac negative. If you then purify the recombinant plasmid
and cut it with EcoRI you would expect to get two fragments, one of 2.7 kb
representing the vector and one of 1 kb representing the genomic DNA
insert. Equally, if you used one of the other restriction enzymes from the
multiple cloning site, for instance HindIII, you would expect to get one
band of 3.7 kb, provided there were no HindIII sites present in the cloned
genomic fragment. However, if HindIII cuts in the genomic DNA insert you
would get more than one fragment, but the sizes of the fragments should