The Applications of Recombinant DNA

RECOMBINANT DNA
There are many different traits that can be introduced to organisms to change their
properties. The following table shows examples of modified traits using cloned
genes and their applications:
MODIFIED TRAIT GENE RECIPIENT APPLICATION (FIELD)
MODIFICATION ORGANISM
Insulin Production Insertion of Bacteria (Medicine)
Human Insulin Production of Human
Gene Insulin in Bacteria
Pest Resistance Insertion of Bt- Corn / Maize (Agriculture)
toxin gene Production of corn
plants with increased
resistance to corn
boxer
Delayed Ripening Disruption of a Tomato plant Agriculture)
gene for a ripening Production of plants
enzyme (e.g. with fruits that have
polygalacturonase delayed ripening
) fruits. These fruits
will
survive longer
transport time,
allowing their
delivery to further
locations (i.e. export
deliveries)
Chymosin Insertion of a gene Bacteria (Industry)
Production for chymosin Enhance large scale
production of
chymosin. This
enzyme
serves as a substitute
for rennet in the
coagulation of milk.
Rennet has to be
harvested from
calves. The large
scale production of
this enzyme in
bacteria provides an
abundant supply of
this important
component for the
cheese production
industry.
PCR Amplification
Once a desired trait is chosen, information must be acquired for either its detection
or expression in a given organism.
1. Detection
 Some researchers may be interested in determining if a given gene/trait is
available in a particular organism. If no previous research provides this
information, researchers may test the DNA of different organisms for the
presence of these specific genes. A technique that allows the detection of
specific genes in target organisms is called PCR.
 PCR amplification is an in-vitro method that simulates DNA replication in vivo.
It utilizes a thermostable (heat-resistant) DNA polymerase that builds single
stranded DNA strands unto unwound DNA templates.

 PCR uses repeated cycles of incubation at different temperatures to promote

the unwinding of the DNA template (~95°C); the annealing of a primer (a
~20bp oligonucleotide sequence (recall RNA primers in DNA replication) onto
the ssDNA template strand (~54 - 60°C); and the extension of the generated
ssDNA strand through the binding of complementary bases to the template
strand (~72° C). The thermostability of the polymerase allows it to survive the
repeated cycles of denaturation, annealing and extension with little loss of
enzyme function. Each cycle of PCR doubles the amount of the target
sequence. A typical PCR experiment uses about 35 cycles of amplification.
This increases the original amount of the target sequence by 235 (i.e. ~34
billion) times.

 Gene detection by PCR involves the design of primers that would only bind to
sequences that are specific to a target. For example, researchers would want
to find out if gene X (e.g. the gene for insulin) is available in a target organism
(e.g. a mouse, Mus musculus). Primers may be designed by looking at the
available sequences for gene X in the databases (e.g. all the genes for insulin
in different organisms; humans, pigs, cows, etc.). The different gene X
sequences must be aligned/ compared to match areas of sequence similarity
(conserved sequences) and areas of sequence dissimilarity (non-conserved
sequences). Primers designed to have the same sequence as the conserved
areas will be specific for binding gene X sequences in all the target
organisms. Primers designed to have the same sequence as the non-
conserved areas will only be specific for the organisms which match its
sequence.