Biology Reference
In-Depth Information
The M13 phage does not contain excess DNA that can be removed. However,
there is a 507-bp region that contains the origins of replication for the viral DNA
and its complementary strand. In most vectors derived from M13, foreign DNA is
inserted at this site. Polylinkers and the
lacZ
gene have been inserted into M13
vectors so that white plaques are formed instead of blue if exogenous DNA is
inserted into the
lacZ
cloning site.
6.3.4 Phagemids
A phagemid is an engineered vector that contains plasmid and M13 compo-
nents. Phagemids provide another method for obtaining ss DNA. Phagemids
carry two replication origins, one a standard plasmid origin and the other
derived from M13. The M13 origin is crucial for the synthesis of ss DNA.
However, production of ss DNA requires enzymes and coat proteins coded by
phage genes, which are lacking in phagemids. As a result, cells containing a
phagemid vector must be coinfected with a helper phage if ss DNA is desired.
The helper phage converts the phagemids into ss DNA molecules that are then
assembled into phage particles and secreted from the cell.
6.3.5 BACs
Bacterial artificial chromosomes
(BACs) were developed so that scientists could
insert very large segments of DNA and have it cloned in
E. coli
. BACs can incor-
porate an average of 140kb of exogenous DNA and are based on the
E. coli
F-factor plasmid; the
E. coli
F-factor plasmid maintains a low copy number in
bacterial cells, thus minimizing the possibility of recombination that could result
in the production of chimeric clones (
Shizuya et al. 1992
). Basic BACs do not con-
tain a selection system or reporter genes suitable for expression in eukaryotic
cell lines. However, BACs have been modified as expression vectors in eukaryotic
cells (
Kim et al. 1998
).
BACs were extensively used in the Human Genome Project, a project that used
Sanger sequencing methods (see Chapter 7). The advantages of the BAC vector
are that it can maintain up to 300 kb of DNA, and the clones are highly stable in
their host even after 100 generations. BACs thus facilitate the construction of
DNA libraries of complex genomes because they allow fuller representation of
all sequences.
6.4 cDNA Cloning
cDNA is DNA that is
complementary
to the mRNA that is transcribed from
the gene. Because the mRNA is processed, it will lack introns and regulatory
