Recombinant DNA technology, joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. Since the focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Although it is relatively easy to isolate a sample of DNA from a collection of cells, finding a specific gene within this DNA sample can be compared to finding a needle in a haystack. Consider the fact that each human cell contains approximately 2 metres (6 feet) of DNA. Therefore, a small tissue sample will contain many kilometres of DNA. However, recombinant DNA technology has made it possible to isolate one gene or any other segment of DNA, enabling researchers to determine its nucleotide sequence, study its transcripts, mutate it in highly specific ways, and reinsert the modified sequence into a living organism.
DNA Cloning
In biology a clone is a group of individual cells or organisms descended from one progenitor. This means that the members of a clone are genetically identical, because cell replication produces identical daughter cells each time. The use of the word clone has been extended to recombinant DNA technology, which has provided scientists with the ability to produce many copies of a single fragment of DNA, such as a gene, creating identical copies that constitute a DNA clone. In practice the procedure is carried out by inserting a DNA fragment into a small DNA molecule and then allowing this molecule to replicate inside a simple living cell such as a bacterium. The small replicating molecule is called a DNA vector (carrier). The most commonly used vectors are plasmids (circular DNA molecules that originated from bacteria), viruses, and yeast cells. Plasmids are not a part of the main cellular genome, but they can carry genes that provide the host cell with useful properties, such as drug resistance, mating ability, and toxin production. They are small enough to be conveniently manipulated experimentally, and, furthermore, they will carry extra DNA that is spliced into them.
Processes of Recombinant DNA Technology
Recombinant DNA (rDNA) technology refers to the process of joining DNA molecules from two different sources and inserting them into a host organism, to generate products for human use. Can you put the DNA molecules in the host organism first and then cut and join them? No! This process involves multiple steps that have to proceed in a specific sequence to generate the desired product. Let’s understand each step in detail.
1. Isolation of Genetic Material
We already know that the genetic material of all living organisms is ‘nucleic acid’. In most organisms, it is DNA, whereas in some it is RNA. The first step in rDNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules.
However, in a normal cell, the DNA not only exists within the cell membrane, but is also present along with other macromolecules such as RNA, polysaccharides, proteins, and lipids. So, how do we break open the cell and obtain DNA that is free from other macromolecules? We can use the following enzymes for specific purposes:
- Lysozyme – to break bacterial cell wall.
- Cellulase – to break plant cell wall.
- Chitinase – to break fungal cell wall.
- Ribonuclease – removes RNA.
- Protease – removes proteins (such as histones that are associated with DNA).
Other macromolecules are removable with other enzymes or treatments. Ultimately, the addition of ethanol causes the DNA to precipitate out as fine threads. This is then spooled out to give purified DNA.
Steps in DNA isolation [Source: Wikimedia Commons]
2. Restriction Enzyme Digestion
Restriction enzymes act as molecular scissors that cut DNA at specific locations. These reactions are called ‘restriction enzyme digestions’. They involve the incubation of the purified DNA with the selected restriction enzyme, at conditions optimal for that specific enzyme.
The technique – ‘Agarose Gel Electrophoresis’ reveals the progress of the restriction enzyme digestion. This technique involves running out the DNA on an agarose gel. On the application of current, the negatively charged DNA travels to the positive electrode and is separated out based on size. This allows us to separate and cut out the digested DNA fragments. The vector DNA is also processed using the same procedure.
Image of digested DNA after agarose gel electrophoresis [Source: Wikimedia Commons]
3. Amplification Using PCR
Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA sequence using the enzyme – DNA polymerase. It helps to amplify a single copy or a few copies of DNA into thousands to millions of copies. PCR reactions are run on ‘thermal cyclers’ using the following components:
- Template – DNA to be amplified
- Primers – small, chemically synthesized oligonucleotides that are complementary to a region of the DNA.
- Enzyme – DNA polymerase
- Nucleotides – needed to extend the primers by the enzyme.
Thermal Cycler [Source: Wikimedia Commons]
The cut fragments of DNA can be amplified using PCR and then ligated with the cut vector as explained below.
4. Ligation of DNA Molecules
The purified DNA and the vector of interest are cut with the same restriction enzyme. This gives us the cut fragment of DNA and the cut vector, that is now open. The process of joining these two pieces together using the enzyme ‘DNA ligase’ is ‘ligation’. The resulting DNA is ‘recombinant DNA‘.
Restriction enzyme digestion followed by ligation. [Source: Wikimedia Commons]
5. Insertion of Recombinant DNA Into Host
In this step, the recombinant DNA is introduced into a recipient host cell. This process is ‘Transformation’. Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make them ‘competent’ to accept new DNA. (The topic – Tools of Biotechnology explains a few ways to make cells competent).
During transformation, if a recombinant DNA bearing a gene for ampicillin resistance is transferred into recipient E. coli cells, then the E. coli cells also become ampicillin-resistant. This aspect is useful in differentiating transformed cells from non-transformed cells.
For example, if we spread the transformed cells on agar plates containing ampicillin, only the transformed, ampicillin-resistant cells will grow while the untransformed cells will die. Therefore, in this case, the ampicillin resistance gene acts as the ‘selectable marker’.
6. Obtaining Foreign Gene Product
The recombinant DNA multiplies in the host and is expressed as a protein, under optimal conditions. This is now a recombinant protein. Small volumes of cell cultures will not yield a large amount of recombinant protein. Therefore, large-scale production is necessary to generate products that benefit humans. For this purpose, vessels called bioreactors are used.
Bioreactors are large containers with a continuous culture system, where the fresh medium is added from one side and used medium is taken out from another side. Bioreactors can process about 100-1000 litres of cell cultures. A bioreactor provides optimum conditions (temperature, oxygen, pH, vitamins etc.) to biologically convert raw materials into specific proteins, enzymes etc.
‘Stirred-tank bioreactor’ is the most common type of bioreactor. It is usually cylindrical and has the following parts:
- Agitator system – to stir the contents evenly.
- Oxygen delivery system – to introduce air into the system.
- Foam control system
- Temperature control system
- pH control system
- Sampling ports – to take out small amounts of culture.
Bioreactor [Source: Wikimedia Commons]
7. Downstream Processing
Before the protein is marketed as a final product, it is subjected to downstream processing which includes:
- Separation and purification.
- Formulation with suitable preservatives.
- Clinical trials to test the efficacy and safety of the product.
- Quality control tests.
Application of recombinant DNA technology
- DNA technology is also used to detect the presence of HIV in a person.
- Application of recombinant DNA technology in Agriculture – For example, manufacture of Bt-Cotton to protect the plant against ball worms.
- Application of medicines – Insulin production by DNA recombinant technology is a classic example.
- Gene Therapy – It is used as an attempt to correct the gene defects which give rise to heredity diseases.
- Clinical diagnosis – ELISA is an example where the application of recombinant
