PROCESS OF RECOMBINANT DNA TECHNOLOGY

Recombinant technology is a complicated process. Several steps lead to the desired goal. The major steps are:

(1) Isolation of DNA,

(2) Digestion of DNA by restriction endonuclease enzyme,

(3) Gene amplification,

(4) Introduction of recombinant DNA into host cells,

(5) Identification of recombinants,

(6) Gene product manufacture, and

(7) Processing.

(1) Isolation of DNA: Isolation of the Genetic Material (DNA)

Nucleic acid (DNA or RNA) is the genetic material of all organisms. It is DNA in majority of 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), cellulase (plant cells), chitinase (fungus).

As we know that genes are present on long molecules of DNA intertwined with proteins like histones, the RNA can be removed by treating with ribonuclease 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 suspension.

(2) DNA digestion by restriction enzymes

The vector and the target DNA fragment can be separately digested with the same restriction enzyme.

The digested vector and the target DNA fragment are then incubated together in the presence of DNA ligase enzyme.

Incubation results in bonding two types of DNA by phosphodiester bonds between them.

Thus, deoxyribose-phosphate backbones of vector molecule and the target DNA fragment are covalently linked, forming a recombinant DNA molecule.

Another possibility in this experiment is the rejoining of the sticky ends of the vector molecule itself, forming a circular vector DNA molecule that is without foreign DNA molecule.

This possibility is eliminated by treating digested, vector with alkaline phosphatase or by using different restriction enzymes.

(3) Gene amplification

This is the process of selective multiplication of a specific region of DNA molecule.

The process has also been used to produce DNA fragments for cloning.

Amplification is achieved by a special method known as polymerase chain reaction (PCR) developed by Kary Mullis in 1985 for which he shared 1993 Nobel Prize.

The principle underlying the technique is to heat double stranded DNA molecule to a high temperature so that the two DNA strands separate into 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) Primers : Primers, which are oligo-nucleotides, usually 10-18 nucleotides long, that hybridize to the target DNA region, one to each strand of the double helix. Two primers are required and 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 (>90º) is required to carry out the synthesis of new DNA. The polymerase which is generally used in PCR reactions is known as Taq polymerase (isolated from a bacterium Thermus aquaticus). Other thermostable polymerases can also be used.

A schematic representation of the three steps performed during PCR. Note that the two primers used are complementary to the 3' end sequences of DNA segment to be amplified

Working Mechanism of PCR :

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

(a) Denaturation: In the denaturation step, the target DNA is heated to a high temperature (usually 94°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: 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 depending on the length and sequence of the primers.

(c) Primer Extension (Polymerisation) : The final step is extension, wherein Taq DNA polymerase (of a thermophilic bacterium Thermus acquaticus) 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. As a result, from a single template molecule, it is possible to generate 2n molecules after n number of cycles.

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PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely.

PCR assays can be performed directly on genomic DNA samples to detect translocation specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.

PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models.

The basis of PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

Viral DNA can likewise be detected by PCR.

The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome.

The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease.

Such early detection may give physicians a significant lead in treatment.

The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques.

Application of PCR

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

(i) Diagnosis of Pathogens : Pathologists use techniques based on detecting specific enzymes or antibodies against disease-related proteins. But these techniques cannot be used for detecting infectious agents that are difficult to culture or that persist at very low levels in infected cells. To overcome these problems, PCR-based assays have been developed that detect the presence of gene sequences of the infectious agents.

(ii) Diagnosis of specific Mutation : PCR can be used to detect the presence of a specific mutation that is responsible for causing a particular genetic disease before the actual onset of the disease. By using PCR, phenylketonuria, muscular dystrophy, sickle cell anaemia, AIDS, hepatitis, chlamydia and tuberculosis can be diagnosed.

(iii) DNA Finger printing: PCR is of immense value in generating abundant amount of DNA for analysis in the DNA fingerprinting technique used in forensic science to link a suspect's DNA to the DNA recovered at a crime scene.

(iv) Detection of Specific Microorganisms: PCR is also used for detecting specific microorganisms from the environment samples of soil, sediments and water.

(v) In Prenatal Diagnosis: It is useful to detect genetic disease in foetus before birth. If the disease is not curable, abortion is recommended.

(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.

Table-II : Comparison between PCR and Gene Cloning

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Definition of Ligase chain reaction (LCR)

A method of DNA amplification similar to PCR.

LCR differs from PCR because it amplifies the probs molecule rather than producing amplicon through polymerization of nucleotides.

Two probes are used per each DNA strand and are ligated together to 'form' a single probe.

LCR uses both a DNA polymerase enzyme and a DNA ligase enzyme to drive the reaction.

Like PCR, LCR requires a thermal cycler to drive the reaction and each cycle results in a doubling of the target nucleic acid molecule. LCR can have greater specificity than PCR.

Polymerase Chain Reaction (PCR) : Technique for obtaining multiple copies of DNA

(4) Introduction of recombinant DNA into host cells:

Once the recombinant DNA molecule has been multiplied, it needs to be inserted into host cells.

Many methods for introduction are available.

Selection of a method depends upon type of vector and the host cell along with other things.

Some common methods are outlined below-

(a) Transformation: This is a method where cells take up DNA from their surroundings. Since, many cells such as those of E.coli, yeast, mammalian cells, etc. do not naturally absorb DNA, hence they need to be made competent. Mandel and Higa (1970) observed that E. coli cells can be made competent to take up external DNA by suspending them in cold calcium chloride.

Transformation in bacterial cell

(b) Transfection: In this method DNA is mixed with charged substances like calcium phosphate, cationic liposomes etc. These are spread on the recipient host cells. Calcium ions carry foreign DNA and release it inside the cell since calcium gets precipitated in the form of calcium phosphate, thus transferring the DNA by endocytosis.

(c) Microinjection and macroinjection: Specially designed micromanipulator is used to inject DNA into cytoplasm or the nucleus of a recipient cell or protoplast. The method is used for direct introduction of DNA into plant or animal cells without using special eukaryotic vectors.

(d) Microprojection (biolistics or particle gun) : Tungsten or gold particles (microparticles) coated with DNA are accelerated to a very high initial velocity. These microprojectiles are carried by other (nylon) microprojectiles or the bullet, causing them to penetrate the cell walls of intact target cells or tissues.

Microinjection : The procedure used for microinjection of DNA.
Microprojectile gun : Schematic representation of microprojectile gun

(e) Electroporation: Short electrical impulses of high field strength are given. These increase the permeability of protoplast membrane by creating transient microscopic pores, thus making the entry of DNA molecules into the cells much easier.

(f) Ti plasmid based gene transfer: A more common method of introducing foreign DNA into plant cells is to use the bacterium Agrobacterium tumefaciens and its Ti plasmid.

This gram negative soil bacterium is a plant pathogen and produces crown gall disease in many dicotyledons including grapes, stone fruits, roses, tomato, sunflower, cotton, soyabean, etc.

Most strains of this bacterium carry tumour inducing (Ti) plasmid.

In nature, Agrobacterium attaches to the leaves of the plants and Ti plasmid is transferred into plant cells.

The plasmid becomes incorporated into plant chromosomal DNA.

Therefore, Ti plasmid has been used as a vehicle for introduction of recombinant DNA into plant cells.

Ti plasmids cause tumours in plants.

Strains of the bacterium have been developed which do not have tumour inducing genes.

However, T region of plasmid plays an important role in gene transfer.

This specific segment of bacterial plasmid DNA is called T-DNA (transferred DNA).

T-DNA has a cloning site into which foreign DNA (DNA insert) is inserted.

This recombinant plasmid is now introduced into the bacterium Agrobacterium tumefaciens.

It is then used to infect cultured cells.

The T region of the plasmid with foreign DNA (or DNA insert) is transferred to plant cells.

It gets integrated with chromosomal DNA of the cell.

Cultured cells are induced to grow into plantlets.

These are planted into the soil where the mature plants are formed.

A-F : Ti plasmid based gene transfer

(5) Identification of recombinant :

After the insertion of recombinant DNA into the host cell, these need to be identified from those which do not possess it.

The methods used to do so consider expression or non-expression of certain characters especially antibiotic resistance-gene (e.g., ampicillin resistance gene) on plasmid vector.

Selectable marker usually provides resistance against a substrate which when added to the culture medium, inhibits the growth of normal cells or tissues in culture, so that only transformed tissues will grow.

Selection of transgenic cells

Thus the simplest method for identification is to grow transformed host cells (with ampicillin resistance gene) on medium containing ampicillin.

This would enable the cells containing this transformed plasmid to grow and form colonies.

There are other methods for detection of recombinants based on the fact that the cloned DNA fragment disturbs the coding sequence of gene.

This is known as insertional inactivation.

Let us consider a plasmid containing genes resistant for two different antibiotics, i.e., ampicillin and tetracycline.

If the target DNA fragment is inserted in a site located in ampicillin resistance gene, this gene will then be inactivated.

Thus, host cells with such a recombinant plasmid will be sensitive to ampicillin but resistant to tetracycline.

These host cells will die when grown on ampicillin containing medium but would grow on medium containing tetracycline.

Self ligated or religated (non-recombinant) vectors would grow on medium containing both ampicillin and tetracycline being resistant to them.

Another, but similar method involves insertional inactivation of lac Z gene.

It is known as blue-white selection, being colour based.

(6) Gene product manufacture:

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 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 and 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 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 lag exponential phase (Lag phase -no significant increase of the cells, exponential phase -rapid multiplication of the cells).

This type of culturing method produces a larger biomass to get higher yields of desired protein.

Small volume cultures cannot give large quantities of the products.

To produce large quantities of these products, development of "bioreactors" was required where large volumes (100-1000 litres) of culture can be processed, Hence, bioreactors are like vessels in which raw materials are biologically converted into specific products, individual enzymes using microbial, plant, animal or human cells.

A bioreactor provides the optimal conditions for obtaining the desired product by providing optimum growth conditions such as substrate, temperature, pH, vitamins, oxygen and salts.

One of the most commonly used bioreactor is of stirring type.

The presence of stirrer makes mixing possible and also makes oxygen available through the reactor.

A bioreactor also has an agitatory system, an oxygen delivery system, a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

A. Simple stirred tank bioreactor. B. Sparged stirred tank bioreactor through
which sterile (free from any germs) air bubbles are sparged.

(7) Downstream Processing :

Once the product is ready, it has to be processed for commercial use.

This requires purification and strict quality control to maintain the efficacy.

The products based on biotechnology must ensure that they satisfy the consumer needs and are not harmful.

Therefore, a thorough checking of products at each level of manufacture is done.

The manufacturing process and the quality control methods vary with each product.