Download Recombinant DNA technology

The overall goal of recombinant-DNA
technology is to identify, isolate. manipulate.
and re-express genes from a given host
 Some of the practical goals of such cut-andpaste technology is to:
1) develop a basic understanding of the function
and regulation of known gene products.
2) identify new genes whose protein products
have not been isolated .
3) correct endogenous genetic defects
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4)express foreign genes in disease-susceptible
hosts
5) manufacture large quantities of a protein
product for widespread use
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DNA is composed of two antiparallel strands
bound by hydrogen bonds between their
nitrogenous-base side chains
Genetic information is provided by the purine and
pvrimidine bases which are linked to a sugar
phosphate backbone.
This structural support is a series of deoxyribose
residues linked by phosphodiester bonds, which
are created by covalently joining a hydroxvl group
at the 3-carbon position on one deoxyribose
residue with a phosphate group located on the 5’
carbon of an adjacent sugar group.
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RECOMBINANT DNA TECHNOLOGY:
A SERIES OF PROCEDURES USED TO JOIN
TOGETHER (RECOMBINE)DNA SEGMENTS.
A RECOMBINANT DNA MOLECULE IS
CONSTRUCTED (RECOMBINED) FROM SEGMENTS
FROM 2 OR MORE DIFFERENT DNA MOLECULES.
UNDER CERTAIN CONDITIONS, A RECOMBINANT
DNA MOLECULE CAN ENTER A CELL AND
REPLICATE THERE, AUTONOMOUSLY (ON ITS
OWN) OR AFTER IT HAS BECOME INTEGRATED
INTO A CHROMOSOME.
An over view
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Treat DNA from both sources with the same restriction
endonuclease (BamHI in this case).
BamHI cuts the same site on both molecules
5' GGATCC 3'
3' CCTAGG 5'
The ends of the cut have an overhanging piece of
single-stranded DNA.
These are called "sticky ends" because they are able to
base pair with any DNA molecule containing the
complementary sticky end.
In this case, both DNA preparations have
complementary sticky ends and thus can pair with
each other when mixed.
DNA ligase covalently links the two into a molecule of
recombinant DNA.
To be useful, the recombinant molecule must
be replicated many times to provide material
for analysis, sequencing, etc.
Producing many identical copies of the same
recombinant molecule is called cloning.
Cloning can be done in vitro, by a process
called the polymerase chain reaction (PCR).
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The use of cloning is interrelated with
Recombinant DNA in classical biology, as the
term "clone" refers to a cell or organism derived
from a parental organism, with modern
biology referring to the term as a collection of
cells derived from the same cell that remain
identical.
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IN THE CLASSICAL INSTANCE, THE USE OF
RECOMBINANT DNA PROVIDES THE INITIAL CELL
FROM WHICH THE HOST ORGANISM IS THEN
EXPECTED TO RECAPITULATE WHEN IT
UNDERGOES FURTHER CELL DIVISION, WITH
BACTERIA REMAINING A PRIME EXAMPLE DUE TO
THE USE OF VIRAL VECTORS IN MEDICINE THAT
CONTAIN RECOMBINANT DNA INSERTED INTO A
STRUCTURE KNOWN AS A PLASMID.
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Plasmids are extrachromosomal self-replicating
circular forms of DNA present in most bacteria,
such as Escherichia coli (E. Coli), containing
genes related to catabolism and metabolic
activity, and allowing the carrier bacterium to
survive and reproduce
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These genes represent characteristics of
resistance to bacteriophages and antibiotics and
some heavy metals, but can also be fairly easily
removed or separated from the plasmid by
restriction endonucleases, which regularly
produce "sticky ends" and allow the
attachment of a selected segment of DNA,
which codes for more "reparative" substances,
such as peptide hormone medications
including insulin, growth hormone, and
oxytocin.
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Diabetic patients, whose elevated sugar levels
(see fig. 1) are due to impaired insulin
production, have been treated with insulin
derived from the pancreas glands of abattoir
animals. The hormone, produced and secreted
by the beta cells of the pancreas' islets of
Langerhans,(2) regulates the use and storage of
food, particularly carbohydrates.
Humulin is synthesized by inserting the
insulin gene into a suitable vector, the
E. coli bacterial cell, to produce an
insulin that is chemically identical to its
naturally produced counterpart.
This has been achieved using
Recombinant DNA technology.