Processes of Recombinant DNA Technology

Processes of Recombinant DNA technology

 

Recombinant DNA technology, also known as genetic engineering, involves manipulating DNA to create new combinations of genetic material. Here's a detailed step-by-step process:

 

  • Isolation of Genetic Material: The first step is to isolate the desired DNA in its pure form, free from other macromolecules. This is done by treating plant or animal cells with specific enzymes such as cellulase, lysozyme, and chitinase to break down the cell walls and membranes, allowing for the extraction of pure DNA.DNA should be separated from its histone proteins and RNA.  This can be carried out by using the enzymes ribonuclease for RNA and proteases for histone proteins. Finally, pure DNA precipitates following the addition of chilled ethanol.
  • Cutting of DNA at Specific Location -To observe the development of a restriction enzyme digestion, cutting of DNA at a specific place is carried out using a restriction enzyme and Agarose gel electrophoresis. After cutting the source DNA and vector DNA with a particular restriction enzyme to remove the 'gene of interest'.
  • Amplification of Gene of Interest using PCR - PCR, or Polymerase Chain Reaction, is a revolutionary technique in molecular biology that allows for the amplification of a specific segment of DNA, including genes of interest. The reaction mixture for PCR typically consists of the Double-stranded DNA Fragment (gene of interest, Primers (small chemically synthesized oligonucleotides that are complementary to the region of this DNA, DNA polymerase (isolated from bacterium, Thermus aquaticus) that does not denature and remains active even at high temperature.

PCR involves a series of temperature-controlled reactions carried out in a thermal cycler, a machine that can precisely control temperature changes. The process typically consists of three main steps:

 

    1. Denaturation: The DNA sample containing the gene of interest is heated to near-boiling temperatures (around 94-98°C). This causes the double-stranded DNA to separate into two single strands, exposing the template for DNA synthesis.

    2. Annealing: The reaction temperature is then lowered to around 50-65°C. Short DNA sequences called primers, which are specifically designed to bind to sequences flanking the target region of the gene of interest, anneal (bind) to their complementary sequences on each of the single-stranded DNA templates.

    3. Extension (Elongation): Once the primers are bound, the temperature is raised again to around 72°C, the optimal temperature for the DNA polymerase enzyme used in PCR. This enzyme extends the primers by adding nucleotides complementary to the template strand, synthesizing new DNA strands. As the DNA polymerase enzyme moves along the template, it synthesizes a complementary strand for each template strand, creating two double-stranded DNA molecules.Amplification of Gene of Interest using PCR - PCR, or Polymerase Chain Reaction, is a revolutionary technique in molecular biology that allows for the amplification of a specific segment of DNA, including genes of interest. The reaction mixture for PCR typically consists of the Double-stranded DNA Fragment (gene of interest, Primers (small chemically synthesized oligonucleotides that are complementary to the region of this DNA, DNA polymerase (isolated from bacterium, Thermus aquaticus) that does not denature and remains active even at high temperature.

       

 

 

These three steps - denaturation, annealing, and extension - are repeated in cycles, typically 20-40 times, depending on the desired amount of amplification. With each cycle, the amount of DNA target sequence doubles, resulting in exponential amplification.

 

  • Insertion of Recombinant DNA into the Host Cell / Organism: There are many methods for transferring the ligated DNA to recipient cells. Transferring a recombinant DNA gene for antibiotic resistance (ampicillin) into E.coli cells results in the host cells becoming ampicillin-resistant. If we spread the transformed cells on ampicillin-containing agar plates, only the transformants will proliferate while the untransformed recipient cells will die. The ampicillin resistance gene in this circumstance is referred to as a selectable marker.
  • Obtaining the foreign gene product – The foreign DNA replicates in plant or animal cells, producing desired protein. Foreign genes are expressed in host cells under optimised conditions to produce recombinant proteins. The recombinant cell is replicated in a continuous culture system, with used medium drained from one side and fresh media fed from the other to keep the cells in their physiological active phase.
  • Bioreactor - Bioreactors are essential tools in recombinant DNA technology for the large-scale production of foreign gene products.Thus, bioreactors may be regarded as tanks in which raw ingredients are biologically transformed into specialised products, enzymes, and so on, using microbial plant, animal, or human cells. A bioreactor creates the ideal growing circumstances for the intended product (temperature, pH, substrate, salts, vitamins, and oxygen).
  • Stirred-Tank Bioreactors: These bioreactors use mechanical or gaseous means to stir the contents, ensuring uniform distribution of nutrients and maintaining homogenous conditions. They are widely used in industrial production of biological products such as enzymes, antibodies, vaccines, and recombinant proteins
  • Downstream Processing: After the production of the desired protein, it undergoes downstream processing, which involves the isolation, purification, and preservation of the final products before it is marketed.