Gene Cloning

Gene Identification and DNA Libraries 95
Head
Tail
Non-essential
region
Lytic
functions
Figure 4.5 Physical map of bacteriophage λ. The regions responsible for
head and tail assembly and also the lytic functions are shown. The regions that
can be deleted without affecting the normal functions (gray stripes) and the
region that can be replaced by cloned DNA (dark blue) are shown.
more DNA than is present in its normal genome. However, parts of the λ
genome can be deleted without affecting the ability of the bacteriophage to
infect host cells and assemble new virus particles (Figure 4.5). Cloning vectors
which have had these regions removed are called insertion vectors,
and they can accommodate about 12 kb of foreign DNA (Figure 4.6a). In
addition these vectors are generally engineered to have a unique restriction
site for a commonly used restriction enzyme such as EcoRI.
A second class of λ phage vectors called replacement vectors (Figure
4.6b) is based on exchange of the non-essential region (Figure 4.5) for the
DNA to be cloned. These vectors can accept larger inserts because they
have had more of the λ DNA removed. Because of the size constraints
placed on successful packaging (Figure 4.4), these vectors contain a stuffer
fragment making them large enough for replication and packaging. This
stuffer fragment is removed during cloning and replaced with the insert
DNA. Using λ replacement vectors it is possible to clone fragments
between 9 and 23 kb.
Bacteriophage λ DNA can be manipulated in vitro in the same way as
plasmid DNA. It can be cut with restriction enzymes and ligated with
genomic DNA similarly treated to give recombinants, which can be used in
the construction of a library. It is possible to introduce these recombinant
molecules into E. coli by transformation where they will direct the host to
produce phage particles. This approach, however, fails to take account of
one of the most useful features of phage λ, namely its ability to introduce
DNA into E. coli with a high frequency by its normal infection process.
4.11 Packaging Bacteriophage λ In Vitro
Because bacteriophage will essentially self-assemble, it is possible to package
DNA into virus particles in the test tube and then to use these to infect
E. coli. This process, referred to as in vitro packaging, can yield about 10 9
plaques per mg of DNA. Mixing concatemeric recombinant DNA with viral
head precursors, phage tails and other proteins required for packaging
results in mature viral particles containing recombinant DNA, which can
be used to infect E. coli cells and which will produce plaques on a bacterial