Biomedical Engineering Reference
In-Depth Information
CHAPTER5
Cosmids, phasmids and
other advanced vectors
along the genome to isolate a gene, and this topic is
covered in Chapter 6. In many instances, the desired
gene will be relatively easy to isolate and a simpler
cloning vector can be used. Once isolated, the cloned
gene may be expressed as a probe sequence or as a
protein, it may be sequenced or it may be mutated
in vitro
. For all these applications, small specialist
vectors are used.
Introduction
In the 1970s, when recombinant DNA technology
was first being developed, only a limited number
of vectors were available and these were based on
either high-copy-number plasmids or phage
. Later,
phage M13 was developed as a specialist vector to
facilitate DNA sequencing. Gradually, a number of
purpose-built vectors were developed, of which
pBR322 is probably the best example, but the cre-
ation of each one was a major task. Over time, a
series of specialist vectors was constructed, each
for a particular purpose. During this period, there
were many arguments about the relative benefits of
plasmid and phage vectors. Today, the molecular
biologist has available an enormous range of vectors
and these are notable for three reasons. First, many
of them combine elements from both plasmids and
phages and are known as
phasmids
or, if they contain
an M13
ori
region,
phagemids
. Secondly, many differ-
ent features that facilitate cloning and expression
can be found combined in a single vector. Thirdly,
purified vector DNA plus associated reagents can
be purchased from molecular-biology suppliers.
The hapless scientist who opens a molecular-biology
catalogue is faced with a bewildering selection of
vectors and each vender promotes different ones.
Although the benefits of using each vector may be
clear, the disadvantages are seldom obvious. The
aim of this chapter is to provide the reader with a
detailed explanation of the biological basis for the
different designs of vector.
There are two general uses for cloning vectors:
cloning large pieces of DNA and manipulating
genes. When mapping and sequencing genomes,
the first step is to subdivide the genome into man-
ageable pieces. The larger these pieces, the easier it is
to construct the final picture (see Chapter 7); hence
the need to clone large fragments of DNA. Large
fragments are also needed if it is necessary to 'walk'
λ
Vectors for cloning large
fragments of DNA
Cosmid vectors
As we have seen, concatemers of unit-length λ DNA
molecules can be efficiently packaged if the
cos
sites,
substrates for the packaging-dependent cleavage,
are 37-52 kb apart (75 -105% the size of
+
DNA).
In fact, only a small region in the proximity of the
cos
site is required for recognition by the packaging
system (Hohn 1975).
Plasmids have been constructed which contain a
fragment of
λ
DNA including the
cos
site (Collins &
Brüning 1978, Collins & Hohn 1979, Wahl
et al.
1987, Evans
et al.
1989). These plasmids have been
termed
cosmids
and can be used as gene-cloning
vectors in conjunction with the
in vitro
packaging
system. Figure 5.1 shows a gene-cloning scheme
employing a cosmid. Packaging the cosmid recombin-
ants into phage coats imposes a desirable selection
upon their size. With a cosmid vector of 5 kb, we
demand the insertion of 32- 47 kb of foreign DNA -
much more than a phage-
λ
vector can accommod-
ate. Note that, after packaging
in vitro
, the particle
is used to infect a suitable host. The recombinant
cosmid DNA is injected and circularizes like phage
DNA but replicates as a normal plasmid without
the expression of any phage functions. Transfor-
med cells are selected on the basis of a vector drug-
resistance marker.
λ
