Biomedical Engineering Reference
In-Depth Information
Phagemids
Phagemids are plasmids that contain two different origins of replica-
tion. They not only contain their own origin for propagation as a plasmid,
but contain a phage origin of replication as well. When a bacterium that
contains the plasmid is infected with a “helper phage”, the phage ori-
gin on the phagemid becomes active. Single stranded DNA is produced
from the plasmid DNA and secreted into the bacterial broth. Vector pairs
that have the phage origin in opposite direction are available, and as a
result single stranded DNA representing both (+ and -) strands of the
DNA can be generated.
Examples of phagemids include “Bluescript” vectors (pBluescriptl,
pBluescriptll, etc.), which are derived from the pUC19 plasmid.
Cosmids
Standard plasmid vectors cannot replicate if large pieces of foreign
DNA have been cloned into them, and consequently the use of plasmids
is limited to the cloning of DNA inserts that are only a few thousand base
pairs. Cosmids were designed to overcome this limitation, so that larger
pieces of DNA can be cloned. This is particularly useful in the cloning of
intact genes with their corresponding regulatory regions, for example.
Cosmids are plasmids that contain a sequence from the lambda phage
(known as the cos site) which facilitates the cloning of larger DNA frag-
ments, between 30 and 40 kb in size. Cosmids do, however, have their
problems, chief among them the loss of random portions of the inserted
DNA.
Artificial chromosomes
In order to be able to clone even larger stretches of DNA, up to 5000 kb
(5 million base pairs), vectors have been designed that can accommo-
date these large DNA pieces. The large size of these cloned fragments
allows them to retain many of the structural characteristics of native ge-
nomic DNA, including chromatin structure. These cloned fragments of
DNA in these vectors are therefore called artificial chromosomes. Two
types of artificial chromosomes have been developed, yeast artificial
chromosomes (YAC), and bacterial artificial chromosomes (BAC).
A YAC is a plasmid that has been adapted to replicate in yeast.
Yeast plasmids are selected using genes that complement mutations
in a biosynthetic pathway of yeast, rather than using an antibiotic re-
sistance marker typical of bacterial vectors. Any yeast containing the
plasmid will be restored to be able to grow in media that requires the
biosynthetic pathway. A BAC plasmid is similar to a YAC vector but is
one that can be manipulated in bacteria.
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