Recombinant DNA technology
it involves steps in sequence
(1) isolation of the genetic material( DNA)
is carried out in the following steps.
(a) DNA is enclosed within the membranes .To release DNA along with other macromolecules such as RNA, proteins, polysaccharides and lipids, bacterial cells/plants or animals tissue are treated with enzymes.such as lysozyme (bacteria), cellulose (plant cells) , chitinase (fungus).
(b) RNA can be removed by treatment with ribonuclease. Whereas proteins can be removed by treatment with protease.
(c) Other molecules can be removed by appropriate treatment and ultimately purified DNA precipitates out after the addition of chilled ethanol.
This can be seen as collection of fine threads in the suspensions which can be removed by spooling.
(2) cutting of DNA at specific locations is done by using restriction and enzymes. The purified DNA molecules are incubated with the specific restriction enzyme at optimum conditions for the enzyme to act.
(3) Isolation and joining of desired DNA fragment is carried out using agarose gel electrophoresis and DNA ligase respectively.
(4) Amplification of gene of interest using polymerase Chain Reaction(PCR) it is a technique in which multiple copies of specific DNA (gene of interest) sequence are made amplification in vitro.
The technique was developed by Kary Mullis in 1985 who received Nobel Prize in chemistry in 1993.
(a) PCR technique requires.
* A DNA template, which is a double stranded DNA that needs to be amplified.
Primers are small chemically synthesized oligo nucleotides of about 10-18 nucleotides that are complementary to a region of template DNA.
* Enzyme used is DNA Taq polymerase (from a bacterium, thermus aquaticus).
* Deoxyribonucleotides are required for the synthesis of news strand.
(b) Steps in PCR
* Denaturation of double stranded DNA is carried out by applying high temperature of 95 degree Celsius for 15 seconds. Each separated single strand acts as a template for DNA synthesis.
* Annealing is carried out by two sets of primers, which are added in the reaction. they anneal to the 3' end of each separated strand. Primers act as initiator of replication.
* Extension is done by DNA polymerase by adding nucleotides complementary to the template in the reaction.
* A thermostable DNA polymerase is used in the reaction because it can tolerate and remain active during the high temperature induced denaturation induced denaturation of dsDNA.
(c) Three steps are repeated many times to obtain several copies of Desire DNA.
(5) Ligation of DNA fragments into a vector
requires a vector DNA and source DNA.
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| Polymerase chain Reaction (PCR) |
(a) These are cut with the same endonuclease to obtain sticky ends.
(b) Both are then ligated by mixing vector DNA gene of interest and enzyme DNA ligase to from Recombinant DNA.
(6) Insertion of Recombinant DNA into the host cell organism of occurs by several methods, before the which the recipient cells are made competent to receive the DNA.
(a) If recombinant DNA carrying antibiotic resistance gene (e.g. ampicillin) is transformed into E.coli cells, the host cell is transformed into ampicillin resistant cells.
(b) ampicillin in resistant gene can be called a selectable marker.
(c) when transformed cells are grown on Agar plates containing ampicillin, only transformants will grow and others will die.
(7) culturing the host cells the cell containing the foreign gene is culture on an appropriate medium at optimal conditions. The DNA gets multiplied.
(8)Extraction of desired gene product is carried out the following Steps
(a) A protein encoding gene expressed in a heterologous host is called recombinant protein.
(b) cells having genes expressed in a heterologous host is called recombinant protein cells having gene of interest can be grown on small on a large scale.
(c) In small scale cells are grown on cultures and then Expressed protein is extracted and purified by various separation methods.
(d) On large scale cells are grown in a continuous culture systems in which fresh medium is added from one side to maintain cells growth phase and desired protein is collected from the other side.
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| Diagrammatic representation of recombinant DNA Technology |
bioreactors
- These are the large volume (100-1000 L) vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant and animals or human cells.
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| Simple stirred-tank Bioreactor |
(1) It provides optimal conditions for achieving the desired product by providing growth conditions like temperature, pH, substrate, salt, vitamins and oxygen.
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| Sparged stirred-tank Bioreactor |
(2) The Sirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.
(a) The stirrer facilities mixing and oxygen availability throughout the bioreactor.
(b) The most commonly used bioreactor are of stirring type.
(3) In the sparged stirred tank bioreactor sterile air bubbles are sparged to increase the surface area for oxygen transfer.
(4) components of a bioreactor are
(a) An agitator system.
(b) An oxygen delivery system
(c) A foam control system
(d) A temperature control system
(e) pH control system
(f) sampling Ports withdraw culture periodically.
Downstream processing
- After the product formation in biosynthetic stage, They are subjected to a series of processes, collectively referred to as downstream processing.
These include
(1) separation of desired product.
(2) purification of desired products.
(3) formulation with preservatives.
Finally, these products undergo strict quality control testing before being marketed as a finished product.
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