Biology Reference
In-Depth Information
Figure 6.3
Schematic outline of a bacteriophage
λ
replacement vector. A linear molecule contains
the
cos
sites, a left (L) and right (R) arm, and a “stuffer” region with nonessential DNA. The vector is
digested with an appropriate restriction enzyme, the stuffer fragment is removed, and the two arms
anneal. Exogenous DNA that has been cleaved with an appropriate restriction enzyme is added, and
the fragment is ligated in. Exogenous DNA fragments of 18 to 22 kb can be incorporated because
these molecules can be successfully packaged by
in vitro
packaging.
Escherichia coli
is infected with the
λ
, and thousands of individual plaques are produced. Each plaque contains many thousands of rep-
licas (clones) of a single phage containing exogenous DNA.
and replacement.
Insertion vectors
have a single target site at which foreign
DNA can be inserted, whereas
replacement vectors
have a pair of sites defining
a fragment that can be removed and replaced by foreign DNA (
Figure 6.3
).
Once exogenous DNA has been inserted into the
λ
vector, this molecule can
be multiplied (cloned) by inserting it into host
E. coli
cells in one of two ways:
transfection and
in vitro
packaging. Naked
λ
DNA (lacking a protein coat) can
be introduced into
E. coli
cells in a process called transfection.
Transfection
is
the infection of bacteria by viral nucleic acid alone. The efficiency of transfec-
tion is
>
10
4
recombinant clones per microgram of donor DNA. This efficiency
would suffice for the construction of genomic libraries from species with small
genomes. However, larger genomes, such as those of insects, require a more
efficient method of inserting the vector DNA into
E. coli
. The way to increase
efficiency in introducing recombinant
λ
DNA molecules into
E. coli
is called
in
vitro
packaging
. By incorporating the recombinant DNA molecules into phage
protein coats,
E. coli
can be infected much more readily, thereby increasing the