Biology Reference
In-Depth Information
What is significant about cloning with cosmids is that larger DNA fragments,
32-47 kb, can be inserted into the vector and still be packaged.
After
in vitro
packaging, cosmids are used to infect a suitable
E. coli
strain.
Infection of
E. coli
involves injection and circularization of the cosmid DNA, but
no phage protein is produced. Transformed
E. coli
cells are identified on the
basis of their resistance to a specific antibiotic.
Although having a large capacity for DNA fragments is a benefit in cloning
with cosmids, it can be a detriment. If, during a partial digestion with restriction
enzymes, two or more genomic DNA fragments join together in the ligation
reaction, a clone could be created with fragments that were not initially adja-
cent to each other. This situation could be a problem if the researcher is inter-
ested in the relationship between a gene of interest and the DNA surrounding
it. The problem can be overcome by size fractionating the partial digest and
dephosphorylating the foreign DNA fragments to prevent them from ligat-
ing together, but this process makes cosmid cloning very sensitive to the exact
ratio of insert and vector DNAs. If the ratio is imbalanced, vector DNAs could
ligate together without containing any exogenous DNA insert. This outcome is
resolved by treating the vector to two separate digestions, thereby generating
vector ends that are incapable of ligating to each other after phosphatasing.
Commonly used cosmid vectors include the pJB8 and the pcosEMBL fam-
ily. The pcosEMBL family was designed to simplify isolation of specific recom-
binants from cosmid libraries and speed up isolating large regions of complex
genomes in an ordered array of overlapping clones (i.e., chromosome walking,
which is described in Section 6.6). The vectors in this family differ by having dif-
ferent cloning sites and different numbers of
cos
sites. Recombinant cosmids in
our theoretical experiment can be identified by rearing
E. coli
in the presence of
the antibiotic ampicillin (
Figure 6.7
).
6.3.3 Cloning in the Filamentous Phage M13
M13
is a filamentous phage of
E. coli
that contains a circular single-stranded
DNA molecule that is 6407 nucleotides in length. M13 only infects strains of
E. coli
that have
F pili
because the site where this phage adsorbs seems to be at
the end of the F pilus (
Figure 6.8
). Replication of M13 does not result in host-cell
lysis. However, the infected cells grow and divide more slowly and extrude up to
1000 virus particles into the medium.
Replication of M13 phage (which is single-stranded) involves conversion of
the DNA to a double-stranded (ds) or replicative form (RF). The ds RF multiplies
