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Principle of Recombinant Dna Technology

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28 views17 pages

Principle of Recombinant Dna Technology

Uploaded by

fikru
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Recombinant DNA Technology

The Human Genome Project has enabled us to determine


the entire human genome sequence during the past few
years despite its length& complexity.

Thus it has greatly contributed to our understanding of many


genetic diseases and has lead scientists to initial
successes in the treatment of some heritable diseases
using “Gene Therapy”.

Several techniques are used in recombinant DNA


technology, including at least 3 right now.
I. Restriction Endonucleases

Initially, these are bacterial enzymes that fight


viral invasion& restrict them as they cleave the
double-stranded (ds) DNA into smaller
fragments.
These enzymes cut DNA at a specific sequence,
and thus used experimentally to obtain
precisely defined DNA pieces called as
restriction fragments.
Restriction enzymes must first recognize short (4-6 base
sequences) stretches of DNA that must be
palindromic.
palindromic These sequences which differ for each
restriction endonuclease.
There are hundreds of restriction enzymes having
different cleavage specificities. They are commercially
available as valuable analytical reagents.

Generally, there are four classes of Restriction Enzymes


based on their chemical composition, restriction sites,
sequence specificity& co-factors needed.

Example: HgaI belongs to Class II.


5ˊ- G A C G C (N) 5 …3ˊ
3ˊ- C T G C G (N) 10 …5ˊ
II. DNA Cloning

It implies the insertion or the


introduction of a foreign DNA
molecule into a replicating cell
(dividing host cell) leading to its
amplification i.e. production of
too many copies of that foreign
molecule.
The target DNA is first cleaved with a
specific restriction enzyme, and then
joined to a DNA molecule called as
cloning vector to form a hybrid or a
recombinant DNA molecule.

Each recombinant DNA molecule conveys its


inserted DNA fragment into a single host
cell, for example, a bacterium, where it is
replicated (or “amplified” or “cloned”).
The process of introducing foreign DNA into a cell
is called transformation for bacteria and
transfection for eukaryotes.

As the host cell multiplies, it forms a clone in


which every host cell (like a bacterium) carries
copies of the same inserted foreign DNA stretch.
This is why is termed cloning.

The cloned DNA is finally released from its vector


by cleavage with the appropriate restriction
endonuclease.
Vector

It is a molecule of DNA to which the fragment of


foreign DNA of interest to be cloned is joined.
Essential criteria of a vector include:
1) It must be capable of autonomous replication within a
host cell independent of chromosomal division.
2) it must contain at least one specific restriction site for
a commercially available digesting enzyme.
3) it has at least one gene that produces antibiotic
resistance.
resistance This can ensure selection of host cells with
the hybrid DNA.
Commonly used vectors include plasmids and
bacterial and animal viruses(eg. Bacteriophages)

1. Prokaryotic plasmids:
plasmids In addition to the single
circular large chromosome of prokaryotes, there are
normally other small circular extra-chromosomal
DNA molecules called plasmids. Plasmid DNA
undergoes replication that may or may not be
synchronized to chromosomal division.

Plasmids may carry genes that convey antibiotic


resistance to the host bacterium.
Plasmids can be easily isolated from bacterial
cells, their circular DNA cleaved at specific
sites by restriction endonucleases, and
foreign DNA inserted (digested with the same
enzyme). The hybrid plasmid can be
reintroduced into a bacterium.
Bacteria will be grown in the presence
of antibiotics (most commonly
Ampicillin and Tetracyclin), thus
selecting for cells containing the
hybrid plasmids.

Bacterial proliferation will make large


numbers of copies of the plasmid
containing the foreign DNA.
Other vectors .2

In addition to prokaryotic plasmids, new improved


vectors have been developed that more efficiently
accommodate larger DNA fragments like Viruses
(e.g. bacteriophage& retroviruses) and some
artificial constructs.
Example:
Human insulin is nowadays widely used
for treating type 1 diabetes mellitus. It
was the first commercial product
created by recombinant DNA
technology developed by Genentech-
Genentech
in 1982. This replaced the commercial
insulin isolated from pigs and cows that
were used for long time in the last
century with many side effects.

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