Processes of rDNA Technology - Practice Questions & MCQ

• Recombinant DNA technology involves several steps in specific sequence such as:
  • isolation of DNA, 
  • cutting of DNA at Specific Locations 
  • isolation of the desired DNA fragment,
  • ligation of the DNA fragment into a vector, 
  • cloning of DNA
  • transferring the recombinant DNA into the host, 
  • culturing the host cells in a medium at large scale and extraction of the desired product
• Let us examine each of these steps in some detail.
   

Isolation of DNA:

• DNA is present in chromosomes within the cell.
• To isolate DNA, the cell at first is to be broken open by treating the cell with an enzyme (lysozyme for bacteria, cellulose for the plant cell, chitinase for fungus) so that DNA with other macromolecules are released.
• The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. 
• Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. 
• This can be seen as a collection of fine threads in the suspension.

Cutting of DNA at Specific Locations:

• Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme.

Isolation of desired DNA Fragment:

• Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion. 
• DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode). The process is repeated with the vector DNA also.
• The joining of DNA involves several processes. 
• After having cut the source DNA as well as the vector DNA with a specific restriction enzyme, the cut out ‘gene of interest’ from the source DNA and the cut vector with space are mixed and ligase is added. 
• This results in the preparation of recombinant DNA.

Cloning of DNA:

• The recombinant plasmid DNA obtained above is allowed to multiply to form a clone of recombinant DNA. 
• This can be done in the host cell or through PCR.

Insertion of Recombinant DNA into the Host Cell/Organism:

• There are several methods of introducing the ligated DNA into recipient cells. 
• Recipient cells after making them ‘competent’ to receive, take up DNA present in its surroundings. 
• So, if a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cells. 
• If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, untransformed recipient cells will die. 
• Since, due to the ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin. 
• The ampicillin resistance gene, in this case, is called a selectable marker.
   

• When a piece of alien DNA is inserted into a cloning vector and transfer it into a bacterial, plant or animal cell, the alien DNA gets multiplied. 
• In almost all recombinant technologies, the ultimate aim is to produce a desirable protein. 
• Hence, there is a need for recombinant DNA to be expressed. 
• The foreign gene gets expressed under appropriate conditions.
• The expression of foreign genes in host cells involves understanding many technical details.
• After having cloned the gene of interest and having optimized the conditions to induce the expression of the target protein, one has to consider producing it on a large scale.
• If any protein-encoding gene is expressed in a heterologous host, it is called a recombinant protein. 
• The cells harboring cloned genes of interest may be grown on a small scale in the laboratory. 
• The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.
• The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while the fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. 
• This type of culturing method produces larger biomass leading to higher yields of the desired protein.