Biomedical Engineering Reference
In-Depth Information
Cosmid
Restriction
endonuclease
target site
Ap
R
Foreign DNA restriction
fragment
cos
Restriction
endonuclease
DNA ligase (high DNA concentration)
+
+
etc.
+
cos
cos
37-52 kb
Packaging
in
vitro
Absorption + injection
Ap
R
Transducing particle
containing cosmid
recombinant
cos
Select Ap
R
clone
DNA circularizes after
injection
Fig. 5.1
Simple scheme for cloning in a
cosmid vector. (See text for details.)
Cosmids provide an efficient means of cloning
large pieces of foreign DNA. Because of their capa-
city for large fragments of DNA, cosmids are par-
ticularly attractive vectors for constructing libraries
of eukaryotic genome fragments. Partial digestion
with a restriction endonuclease provides suitably
large fragments. However, there is a potential prob-
lem associated with the use of partial digests in
this way. This is due to the possibility of two or
more genome fragments joining together in the
ligation reaction, hence creating a clone containing
fragments that were not initially adjacent in the
genome. This would give an incorrect picture of
their chromosomal organization. The problem can
be overcome by size fractionation of the partial
digest.
