Processes of Recombinant DNA Technology

Recombinant DNA technology is the technique of joining DNA from two different sources and introducing it into a host organism to produce useful products.

• It helps produce useful substances such as insulin, hormones, vaccines, and enzymes.
• It allows scientists to multiply a selected gene in large amounts.
• It supports medicine, agriculture, diagnostics, and research.

The first step is to isolate DNA containing the required gene. Since DNA is enclosed within membranes, the cell has to be broken open. This process is called lysis.

How cells are broken

• Bacterial cells are treated with lysozyme.
• Plant cells are treated with cellulase.
• Fungal cells are treated with chitinase.

Purified DNA is cut at specific sites using restriction enzymes. These enzymes act like molecular scissors. They recognise particular DNA sequences and cut the DNA precisely.

Why this step matters

• It helps remove the desired gene from the source DNA.
• It also allows the vector DNA to be cut at a suitable site.

After cutting, DNA fragments of different lengths are formed. These fragments are separated by gel electrophoresis. DNA molecules move in an electric field through agarose gel.

Principle:

• DNA is negatively charged.
• Therefore, it moves towards the positive electrode.
• Smaller fragments move faster than larger fragments.

Identification of fragments:

• DNA fragments are stained and seen as bands under ultraviolet light.
• The desired DNA band is cut out from the gel and purified.
• This recovery of DNA from the gel is called elution.

A typical agarose gel electrophoresis showing the migration of undigested (lane 1) and a digested set of DNA fragments (lane 2 to 4)

The desired gene is often present in a very small amount. It is therefore amplified using Polymerase Chain Reaction (PCR). PCR produces many copies of the selected DNA segment.

Main requirements for PCR:

• template DNA
• two primers
• DNA nucleotides
• thermostable DNA polymerase

Enzyme used: Taq polymerase, which remains active at high temperatures.

Main steps of PCR:

1. Denaturation – DNA strands separate on heating.
2. Annealing – primers attach to complementary sequences.
3. Extension – Taq polymerase synthesises new DNA strands.

Polymerase chain reaction (PCR): Each cycle has three steps: (i) Denaturation; (ii) Primer annealing; and (iii) Extension of primers

The desired gene is joined with a vector DNA. This joining is done by DNA ligase. The resulting DNA is called recombinant DNA.

About vectors:

• A vector is a carrier DNA molecule.
• It carries the desired gene into the host cell.
• Common vectors include plasmids.

Why ligation is necessary:

• Restriction enzymes cut DNA.
• DNA ligase joins DNA fragments.
• Both actions are essential and must not be confused.

The recombinant DNA is introduced into a suitable host cell. This process is called transformation when bacteria take up the recombinant DNA.

How host cells are made ready:

• Cells are first made competent so that they can take up foreign DNA.
• In bacteria, treatment with calcium ions followed by heat shock facilitates DNA entry into cells.

Other methods that may be used:

• electroporation
• microinjection
• biolistics or gene gun

Once inside the host, the foreign gene may replicate and express itself. The host then begins to produce the desired protein or product. This useful substance is called the foreign gene product.

Example:

Microorganisms carrying a useful human gene can be used to produce therapeutic proteins.

Small-scale production is not sufficient for commercial use. Therefore, the transformed host cells are grown in large vessels called bioreactors. These provide ideal conditions for growth and product formation.

Features of a bioreactor:

• large vessel for culturing cells
• system for mixing or agitation
• oxygen supply
• foam control
• temperature control
• pH control
• sampling ports

Common type: A stirred-tank bioreactor is widely used.

(a) Simple stirred-tank bioreactor; (b) Sparged stirred tank bioreactor through which sterile air bubbles are sparged

The product formed in the bioreactor is not immediately ready for use. It must be separated, purified, and processed. These final steps are together called downstream processing.

It may include:

• separation of product from cells or medium
• purification of the product
• formulation and packaging
• quality testing

• Cells are first broken open using specific enzymes (such as lysozyme for bacteria) to successfully isolate the genetic material.
• The purified DNA is precisely cut at specific locations using restriction enzymes, which act as "molecular scissors" to extract the desired gene.
• The resulting DNA fragments are separated by size using gel electrophoresis, and the specific target sequence is extracted.
• The desired gene is then amplified into millions of copies using the Polymerase Chain Reaction (PCR) technique.
• The amplified gene is joined to a carrier vector using the enzyme DNA ligase to construct a new molecule called recombinant DNA.
• This recombinant DNA is introduced into a chemically treated, competent host cell (such as a bacterium) through a process known as transformation.
• For commercial use, these transformed host cells are cultured on a massive scale inside large, environmentally controlled vessels called bioreactors.
• The final therapeutic product undergoes downstream processing, which involves rigorous separation, purification, and quality testing before packaging.