Biology Reference
In-Depth Information
in tissue culture also can be used as hosts for baculovirus expression vectors (see
Section 6.8).
A multitude of vectors have been developed commercially for cloning. The
choice of an appropriate vector depends upon the goal of your experiment. To
develop a new vector requires an extensive knowledge of the biology and genet-
ics of
E. coli
or other host cell, the plasmid or bacteriophage, and enzymology. It
is impossible to describe all existing vectors and their uses; furthermore, such a
description would become obsolete rather rapidly. Hundreds of vectors are com-
mercially available, with new vectors engineered and advertised regularly.
Basically, a vector is a segment of DNA with an origin of replication that
allows it to be maintained stably after it is introduced into its host cell. Most
vectors contain unique restriction sites in a region of the vector that contains
nonessential genetic information. The unique restriction sites are where exog-
enous DNA fragments can be inserted into the vector. When several cloning
or restriction sites are combined into a single region, it is called a
polylinker
or
multiple-cloning site.
One widely used plasmid vector series is a derivative of plasmid BR322
(pBR322). It was described and its structure illustrated in Chapter 5 (see Figure
5.2). Another plasmid vector series is the pUC group, and these plasmids con-
tain a functional segment of the
E. coli lacZ
gene (see Figure 5.3). Thus,
E. coli
containing this plasmid produces blue colonies if provided with the appro-
priate substrate in the agar medium. If exogenous DNA is inserted into the
cloning site, which is located within the
lacZ
gene, the
lacZ
gene sequence is
disrupted, the gene product is no longer made, and the
E. coli
colonies are col-
orless, allowing the scientist to choose colonies that have been transformed
with the vector that contains exogenous DNA. This is called blue-white selec-
tion. The pUC plasmids produce an increased copy number in
E. coli
that
results in an increased yield of recombinant DNA molecules compared with the
pBR322 plasmid series.
Cloning can be used to produce gene libraries, develop mutated genes for
experiments, provide single-stranded DNA for sequencing, and permit eukary-
otic genes to be translated in
E. coli
, insect tissue culture cells, or lepidopteran
larvae.
Figure 6.1
identifies many of the steps and procedures involved in clon-
ing, but a full description of all the techniques used is beyond the scope of this
chapter.
Commercial companies will provide genomic or complementary DNA (cDNA)
libraries for a fee if you provide the DNA or RNA. The availability of the
