Recombinant DNA (Rdna) Technology Involves The Following Stages

Recombinant DNA (rDNA) technology involves the following six stages:

1. Isolation of the Genetic Material (DNA) 2. Cutting of DNA at Specific Locations 3. Amplification of Gene of Interest Using PCR 4. Preparation and Insertion of Recombinant DNA into the Host Cell/Organism  5. Obtaining the Foreign Gene Product!

1. Isolation of the Genetic Material (DNA):

Nucleic acid (DNA or RNA) is the genetic material of all organisms. It is DNA in majority organisms.

For cutting the DNA with restriction enzymes it needs to be pure and free from other macromolecules. Because the DNA is covered by the membranes, it has to break the cell open to release DNA and other macromolecules like RNA, proteins, polysaccharides and lipids.

It is obtained by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulose (plant cells), chitinase (fungus). As we know that genes are present on long molecules of DNA inter-wined with proteins like histones, the RNA can be removed by treating with ribo-nuclease while proteins can be removed by treating with protease.

Other molecules are removed by proper treatments. The purified DNA finally precipitates out after the addition of chilled ethanol. This is seen as collection of fine threads in the suspension (Fig. 11.10).

2. Cutting of DNA at Specific Locations:

Under the optimal conditions, the purified DNA is cut by the restriction enzyme. Agarose gel electrophoresis is used to check the progress of a restriction enzyme digestion. Since DNA is a negatively charged molecule, it moves towards the positive electrode (anode). This process is also repeated with the vector.

Formation of Recombinant DNA (rDNA):

After the cutting of the source DNA and 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 enzyme is added. This results in the formation of a rDNA or hybrid DNA or chimeric DNA.

3. Amplification of Gene of Interest Using PCR (Fig. 11.11):

The Polymerase Chain Reaction or PCR, as it is commonly called, was originally invented by Kary Mullis in 1985. Kary Mullis shared the Nobel Prize with Michael Smith in Chemistry in 1993. PCR is best defined as the DNA replication in vitro. It results in the selective amplification of a specific region of a DNA molecule and so can also be used to generate a DNA fragment for cloning.

The basic principle underlying this technique is that when a double-stranded DNA molecule is heated to a high temperature, the two DNA strands separate giving rise to single-stranded DNA molecules. If these single-stranded molecules are copied by a DNA polymerase, it would lead to duplication of the original DNA molecule and if these events are repeated many times, multiple copies of the original DNA sequence can be generated. The basic require­ments of a PCR reaction are the following:

(i) DNA Template:

Any source that contains one or more target DNA molecules to be amplified can be taken as template.

(ii) Two Nucleotide Primers:

Primers, which are oligo-nucleotides that hybridize to the target DNA region, one to each strand of the double helix are required. These primers are oriented with their ends facing each other allowing synthesis of the DNA towards one another.

(iii) Enzyme:

DNA polymerase which is stable at high temperatures (> 90°C) is required to carry out the synthesis of new DNA. The DNA polymerase like Taq polymerase is generally used in PCR reactions which are isolated from a bacterium Thermus aquaticus. Other thermostable (temperature remains stable) polymerases can also be used.

Working Mechanism of PCR (Fig. 11.11):

A single PCR amplification cycle involves three basic steps; denaturation, annealing and extension (polymerisation).

(a) Denaturation (Melting of Target DNA):

In the denaturation step, the target DNA is heated to a high temperature (usually 94° to 96°C), resulting in the separation of the two strands. Each single strand of the target DNA then acts as a template for DNA synthesis.

(b) Annealing (Anneal = Join):

In this step, the two oligo-nucleotide primers anneal (hybridize) to each of the single stranded template DNA since the sequence of the primers is complementary to the 3′ ends of the template DNA. This step is carried out at a lower temperature (usually 40° to 60°C) depending on the length and sequence of the primers.

(c) Extension (Polymerisation):

The final step is extension, wherein Taq DNA polymerase (of a thermophilic bacterium Thermus aquaticus) synthesizes the DNA region between the primers, using DNTPs (deoxynucleoside triphosphates) and Mg²⁺. It means the primers are extended towards each other so that the DNA segment lying between the two primers is copied. The optimum temperature for this polymerization step is 72°C.

To begin the second cycle, the DNA is again heated to convert all the newly synthesized DNA into single strands, each of which can now serve as a template for synthesis of more new DNA. Thus the extension product of one cycle can serve as a template for subsequent cycles and each cycle essentially doubles the amount of DNA from the previous cycle.

Application of PCR:

Some of the areas of application of PCR are briefly mentioned here.

(i) Detection of Pathogens: In recent times, PCR is being used in the detection of HIV (Virus of AIDS),

(ii) Diagnosis of Specific Mutation. Mutations are related to genetic diseases. By using PCR phenylketonuria, muscular dystrophy, sickle cell anaemia, hepatitis, chlamydia and tuberculosis can be diagnosed,

(iii) PCR is also used in DNA Fingerprinting,

(iv) Detection of Specific Microorganisms. In recent years, PCR has also found useful for detecting specific microorganisms,

(v) In Prenatal Diagnosis. It is useful to detect genetic disease in foetus before birth,

(vi) Diagnosis of Plant Pathogens. Many diseases of plants can be detected by using PCR. For examples, viroids (associated with apple, grape, citrus, pear, etc.), viruses (like TMV, bean yellow mosaic virus etc), bacteria, mycoplasmas, etc.

(vii) In Palaeontology. PCR is used to clone the DNA fragments from the mummified remains of humans and extinct animals like wooly mammoth and dinosaurs,

(viii) Gene Therapy. PCR proves to be of immense help in monitoring a gene in gene therapy experi­ments.

4. Preparation and Insertion of Recombinant DNA into the Host Cell/Organism (Fig. 11.12):

The vector DNA {e.g., plasmid DNA) and alien (foreign) DNA carrying gene of interest are cut by the same restriction endonuclease to produce complementary sticky ends. This process of cutting DNA by restriction enzymes is called restriction digestion. With the help of DNA ligase enzyme, the complementary sticky ends of the two DNAs are joined (annealing) to produce a recombinant (chimera) DNA (rDNA). The ligase forms new sugar-phosphate bonds to join two DNAs.

Both direct and indirect methods are used to introduce the ligated DNA into the host cells. If a recombinant DNA bearing gene for resistance to antibiotic ampicillin is transferred into E. coli cells, the host cells become transformed into ampicillin resistant cells. If such bacteria are transferred on a culture plate containing the antibiotic ampicillin, only the resistant forms will grow and others will die. The ampicillin resistance gene in this case is called a selective marker.

5. Obtaining the Foreign Gene Product (Fig. 11.12):

When recombinant DNA is transferred into a bacterial, plant or animal cell, the foreign DNA is multiplied. Most of the recombinant technologies are aimed to produce a desirable protein. So there is a need for expression of recombinant DNA.

After the cloning of the gene of interest one has to maintain the optimum conditions to induce the expression of the target protein one should consider producing it on a large scale. If any protein encoding gene is expressed in a heterologous host it is known as a “recombinant protein”. The cells having cloned genes of interest can be grown on a small scale in the laboratory. The cultures may be used for extracting and purifying the desired protein.

The cells can also be multiplied in a continuous culture system where the used medium is passed out from one side and fresh medium is added from the other side to maintain the cells in their physiologically most active log/exponential phase— rapid multiplication of the cells. This type of culturing method produces a larger biomass to get higher yields of desired protein.

Bioreactors (Fermenters):

Bioreactors are considered as vessels in which raw materials are biologically converted into specific products by microbes, plant and animal cells and/or their enzymes. Small volume cultures cannot give large quantities of the products. Large scale production (100 – 1000 litres) of the products is carried out in bioreactors (Fig. 11.13). A bioreactor provides the optimal conditions for obtaining the desired product by providing optimum growth conditions such as temperature, pW, substrate, vitamins, oxygen and salts.

Types of Bioreactors:

The most commonly used bioreactors are of stirring type. Stirring type bioreactors are:

(i) Simple stirred-tank bioreactor and

(ii) Sparged stirred-tank bioreactor as shown in Fig. 11.13. In the sparged bioreactor, sterile air bubbles are sparged. The surface area for oxygen transfer is increased.

Fermentation Process:

Fermenta­tion is the process by which microor­ganisms turn raw material such as glu­cose into products such as alcohol. The term fermentations originally applied only to anaerobic processes but is now used more broadly to include all processes whether aerobic or anaerobic.

All operations are carried out under sterile conditions to avoid contamina­tion of the culture. The product is ei­ther the cells themselves (biomass) or some useful cell product.

Two basic types of fermentation are possible. These are batch fermenta­tion and continuous fermentation. In batch fermentation, the nutrients and microorganisms are put in a closed re­actor and not changed from outside once the fermentation starts, for example, no more nutrients are added.

When nutri­ents are utilized, the product is sepa­rated from microorganisms. In continu­ous fermentation nutrients are replaced as fast as they are used and products are removed as fast as they are made.

Uses:

The stirred-tank bioreactor is well suited for large-scale production of micro-organisms under aseptic con­ditions for a number of days. It can be used easily in research laboratories. Other advantages are an oxygen deliv­ery system, foam control system, a tem­perature control system, pH control system, etc.

Drawbacks:

Drawbacks in this bioreactor are that it is relatively expensive to run it.

6. Downstream Processing:

After the formation of the product in the bioreactors, it undergoes through some processes before a finished product to be ready for marketing. The processes include (a) separation and (b) purification of products which are collectively called the downstream processing. The product is subjected to quality control testing and kept in suitable preservatives. If drugs are to be manufactured such formulation has to undergo through clinical trials. A proper quality control testing for each product is also needed. The downstream processing and quality control test are different from product to product.

Eukaryotic Vehicles:

We have so far described vectors that are suitable for prokaryotic cells especially in E. coli. The vehicles (DNAs) are constructed in various ways, but the DNA of Simian virus 40 (SV 40) or its derivative is most commonly used vehicle for mammalian cells. This vehicle can accept an insert of a length of 4.3 Kb and it does not contain any marker.

Passenger DNA/ Foreign DNA/DNA Insert:

The DNA which is transferred from one organism into another by joining it with the vehicle DNA, is called passenger or foreign DNA. Generally three types of passenger DNAs are used. These are complementary DNA (cDNA), synthetic DNA (sDNA) and random DNA.

1. Complementary DNA (cDNA):

It is synthesized on RNA template (usually mRNA) with the help of reverse transcriptase enzyme discovered by Temin and Baltimore in 1970 and essential nucleotides (Fig 11.14). The DNA is separated from the RNA-DNA complex in the presence of alkaline phos­phatase enzyme. A cDNA strand is formed on the separated single-stranded DNA template with the help of DNA polymerase enzyme.

2. Synthetic DNA (sDNA):

It is synthesized on DNA template or without a template.

Artificial Synthesis of DNA on Template:

Kornberg and his coworkers (1959) pro­duced DNA from deoxyribonucleoside triphosphates in the presence of DNA polymerase enzyme, metal ions and a segment of viral DNA which acts as a primer.

Artificial Synthesis of DNA without a Template:

Hargobind Khorana and his coworkers in 1970, synthesized the gene which was responsible for coding for tyrosine- tRNA of E. coli. The gene had 207 nucleotide pairs.

3. Random DNA:

Small fragments are formed by breaking a chromosome of an organ­ism in the presence of restriction endonucleases.

Measurement of DNA:

DNA in a cell is measured in terms of DNA content in 1C (Gj phase of cell cycle) cells or genome size in a haploid cell.

DNA content in a 1C human cells is 3.2 picograms.

DNA size in a haploid human cell is 3.2 x 10⁹ bp.

Table: Some of the Recombinant Protein Drugs and their Therapeutic Uses.