Biomedical Engineering Reference
In-Depth Information
are designed so that they cannot live outside of laboratory conditions.
Paul Berg shared the Nobel Prize with Gilbert and Sanger in 1980 for
this technological advance.
B.
Plasmid and viral vectors
A vector is a commonly used term for a plasmid or virus that can be
used for cloning, expressing and/or transferring DNA from one organism
to another. The introduction of DNA sequences from an organism (either
prokaryotic or eukaryotic) into a vector is a basic part of recombinant
DNA technology. Cloning vectors, whether they are plasmid or viral vec-
tors, contain restriction sites that permit the ligation of foreign DNA into
the vector without disrupting sequences necessary for the replication
and function of the vector. The type of vector to be used depends on
the size and complexity of the DNA to be cloned. For example, cloning
genomic DNA requires vectors that can incorporate large pieces of DNA
that can only be accommodated by some specialized vectors.
While the recombinant DNA revolution started with the first manipu-
lated cloned DNA in the early 1970's, its genesis really began much ear-
lier. Recombinant DNA technology would not have been possible without
previous studies of bacteria and the investigations into the structure and
regulation of plasmids and viruses that grow in bacteria, known as bac-
teriophages. An understanding of the biology of these DNAs and their
replication cycles was essential for producing cloned recombinant DNA.
Plasmid
A plasmid is a self-replicating extrachromosomal piece of DNA, which
is usually double stranded and circular in nature. Plasmids are natu-
rally found in bacteria where they frequently carry antibiotic resistance
markers used by the host bacterium for survival. The accumulation of
plasmids accounts for much of the antibiotic resistance evident in new
strains of bacteria that are plaguing our hospitals. Plasmids rely on pro-
teins produced by the host bacteria for their replication.
All plasmids used for recombinant DNA work have certain features
in common. First, they must be able to replicate, and to do this they
contain a replication origin where DNA synthesis begins. Second they
must have a selectable marker, so that only the bacteria that contain
the plasmid will grow in culture. In general, these are antibiotic resis-
tance genes. Third, they must have a site into which the DNA of inter-
est can be inserted, without affecting the replication or selection of the
plasmid. This cloning site contains one or more restriction endonucle-
ase cleavage sites that are not contained elsewhere in the plasmid.
Search WWH ::




Custom Search