Biomedical Engineering Reference
In-Depth Information
Artificial chromosomes were initially conceived of and developed for
use in cloning large fragments of DNA to be used in genome sequenc-
ing projects, such as the human genome project. They have now been
adapted for a variety of uses, including the identification of segments
of chromosomes that encode genes which contribute to human dis-
eases. Artificial chromosomes are also used as a tool to isolate closely
linked genes and study their regulation and function in a near normal
setting.
Sequencing vectors
These are vectors that are designed to facilitate DNA sequencing.
Sequencing vectors contain commonly used primer sites that flank the
MCS (SP6 and T7 sequences are common primer sites). In this way,
DNA can be sequenced without knowing anything about the sequence
of the insert DNA. Many sequencing vectors give rise to single stranded
DNA, which is easier to sequence using dideoxy sequencing since it
does not have to be denatured before adding the polymerase. DNA
sequencing was often carried out using the bacteriophage M13 de-
scribed earlier. However, phagemids have largely replaced M13 as the
vectors of choice for sequencing when single stranded templates are de-
sired. Automated sequencing, however, can easily accommodate double
stranded vectors.
Reporter plasmids
These are used to determine if a particular DNA sequence contains
transcriptional control regions, including promoter and/or enhancer ele-
ments. Reporter plasmids contain a cloning site for the DNA of interest
upstream of a reporter gene, a gene whose product can be easily identi-
fied. The reporter gene is expressed when the cloned upstream DNA is
engaged by transcription factors present in the transfected cell. Expres-
sion of the reporter gene implies that transcriptional control regions that
are present in the cloned piece of DNA. Early constructs used chloram-
phenicol acetyltransferase (CAT) as a reporter gene since its enzymatic
activity was easy to monitor. In addition, enzymes whose activity can
be rapidly detected by spectroscopic methods are frequently used in re-
porter assays. Popular reporter genes include luciferase (luc; an enzyme
isolated from the firefly), or The products of these enzymes can be
detected spectroscopically, using bioluminescent or chemiluminescent
methods, most of which are now automated. Most reporter constructs
now contain a gene encoding a “visible” readout, such as green fluores-
cent protein (GFP). GFP is a protein from the Pacific jellyfish, Aequoria
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