Process of Recombinant DNA Technology

• Recombinant DNA technology involves the following steps:

1. Isolation of DNA
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of a desired DNA fragment.
4. Amplification of the gene of interest.
5. Ligation of the DNA fragment into a vector.
6. Insertion of recombinant DNA into the host.
7. Culturing the host cells on a suitable medium at a large scale.
8. Extraction of the desired gene product.
9. Downstream processing of the products as finished product, ready for marketing.

(i) Isolation of the genetic material (DNA)

• 1. RNA and proteins are removed by the treatment of ribonucleges and proteases respatively.
• 2. After several treatments, the purified DNA is precipitated by adding chilled ethanol.
• The bacterial/plant/animal cell is broken down by enzymes to release DNA, along with RNA, proteins, polysaccharides and lipids.
• Bacterial cell is treated with enzyme lysozyme.
• Plant cell is treated with enzyme cellulose.
• Fungal cell is treated with chitinase.

(ii) Cutting of DNA at specific locations

• The DNA is cut using restriction enzymes.
• The purified DNA is incubated, with the specific restriction enzymes at conditions optimum for the enzyme to act.

(iii) Isolation of desired DNA fragment

• Using agarose gel electrophoresis, the activity of the restriction enzymes can be checked.
• Since the DNA is negatively charged, it moves towards the positive electrode or anode and in the process, DNA fragments separate out based on their sizes.
• The desired DNA fragment is eluted out.

(iv) Amplification of gene of interest using PCR

• The Polymerases Chain Reaction (PCR) is a reaction in which amplification of specific DNA sequence is carried out in vitro.
• This technique was developed by Kary Mullis.
• Requirements for PCR:

1. DNA template: The double-stranded DNA that needs to be amplified
2. Primers:Small chemically synthesised oligonucleotides of about 10-18 nucleotides that are complementary to a region of template DNA.
3. Enzyme: Two commonly used enzymes are Taq polymerase (isolated from thermophilic bacterium. Thermus aquaticus) and Vent polymerase (isolated from Thermococcus litoralis).

• PCR is carried out in the following three steps:

1. Denaturation

• The double-stranded DNA is denatured by high temperature of 95^°C for 15 seconds. Each separated single stranded strand now acts as template for DNA synthesis.

2. Annealing

• Two sets of primers are added which anneal to the 3’ end of each separated strand.
• Primers act as initiators of replication.

3. Extension

• DNA polymerase extends the primers by adding nucleotides complementary to the template provided in the reaction.
• A thermostable DNA polymerase (Taq polymerase) is used in the reaction which can tolerate the high temperature of the reaction.
• All these steps are repeated many times to obtain several copies of desired DNA.

(v) Ligation of DNA fragment into a vector

• The vector DNA and source DNA are cut with the same endonuclease to obtain sticky ends.
• These are then ligated by mixing vector DNA, gene of interest and enzyme DNA ligase to form a recombinant DNA.

(vi) Insertion of recombinant DNA into the host cell/organism

• Introduction of ligated DNA into recipient cells occurs by several methods, before which the recipient cells are made competent to receive the DNA.
• If recombinant DNA carrying antibiotic resistance (e.g., ampicillin) is transferred into E. coli cells, the host cell is transformed into ampicillin – resistant cells.
• The ampicillin resistant gene in this case is called a selectable marker.
• On growing transformed cells on agar plates containing ampicillin, only transformants wil grow and others will die.

(vii) Culturing the host cells

• The transformed host cells are grown in appropriate nutrient medium at optimal conditions.
• The DNA gets multiplied and expresses itself to form desired product.

(viii) Extraction of desired gene product

• On small scale, the cells are grown on cultures in laboratory and then the expressed protein is extracted and purified by different separation techniques.
• On large scale, the cells are grown in a continuous culture system in which fresh medium is added from one side to maintain cells in exponential growth phase and the desired protein is collected from the other side.
• Also here larger biomass is produced which leads to high yield.

(ix) Downstream processing

• All the processes to which a product is subjected to before being marketed as a finished product are called downstream processing.
• It includes:

(a) Separation of the product from the reactor.

(b) Purification of the product.

(c) Formulation of the product with suitable preservatives.

(d) Quality control testing and clinical trials in case of drugs.

Bioreactors

• Bioreactors are vessels of large volumes (100 – 1000 litres) in which raw materials are biologically converted into specific products.
• It provides all the optimal conditions for achieving the desired product by providing optimal growth conditions like temperature, pH, substrate, salt, vitamins and oxygen.

• Stirred – tank bioreactors are commonly used bioreactors.
• These are cylindrical with curved base to facilitate proper mixing of the contents.  
• A bioreactor has the following components:

(i) An agitator system

(ii) An oxygen delivery system

(iii) Foam control system

(iv) Temperature control system

(v) pH control system

(vi) Sampling ports to withdraw cultures periodically.

• The stirrer mixes the contents and makes oxygen available throughout the bioreactor.
• Sparged stirred – tank reactor is a stirred type reactor in which air is bubbled.