This MCQ module is based on: Processes of Recombinant Dna Technology
TOPIC 43 OF 64
Processes of Recombinant Dna Technology
🎓 Class 12
Biology
CBSE
Theory
Ch 9 – Biotechnology: Principles and Processes
⏱ ~14 min
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Processes of Recombinant DNA Technology
Recombinant DNA technology involves several steps in a specific sequence: isolation of DNA, fragmentation of DNA by restriction endonucleases, isolation of the desired DNA fragment, ligation of the fragment into a vector, transferring the recombinant DNA into the host, culturing the host cells in a medium at large scale, and extraction of the desired product. This part walks the whole pipeline, from breaking open a cell to filling a vial.
Step 1 — Isolation of the genetic material
Nucleic acid is the genetic material of all organisms without exception, and in the majority of organisms this is deoxyribonucleic acid. In order to cut DNA with restriction enzymes, it must be in pure form, free from other macromolecules.
Since DNA is enclosed within membranes, the cell has to be broken open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and lipids. This is achieved by treating the cells or tissue with wall-digesting enzymes: lysozyme for bacteria, cellulase for plant cells, and chitinase for fungus.
Genes are located on long molecules of DNA intertwined with proteins such as histones, so the unwanted material must now be removed. RNA can be removed by treatment with ribonuclease, whereas proteins can be removed by treatment with protease. Other molecules are removed by appropriate treatments, and purified DNA ultimately precipitates out after the addition of chilled ethanol. It can then be seen as a collection of fine threads in the suspension — visible enough to be lifted out by spooling it onto a rod.
Notice how the enzyme matches the wall. Bacteria are opened with lysozyme, plant cells with cellulase, fungi with chitinase. The goal is identical in all three cases; the tool changes because peptidoglycan, cellulose and chitin are three different materials. Ask what a cell wall is made of and you can predict which enzyme is needed.
Step 2 — Cutting DNA at specific locations
Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme at the optimal conditions for that specific enzyme. Agarose gel electrophoresis is employed to check the progression of the digestion; since DNA is a negatively charged molecule, it moves towards the positive electrode, the anode.
The process is then repeated with the vector DNA also — using the same enzyme, so that the ends will match.
The joining of DNA involves several processes. After cutting the source DNA as well as 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 is added. This results in the preparation of recombinant DNA.
Step 3 — Amplification of the gene of interest using PCR
PCR stands for Polymerase Chain Reaction. In this reaction, multiple copies of the gene or DNA of interest are synthesised in vitro using two sets of primers and the enzyme DNA polymerase.
The primers are small, chemically synthesised oligonucleotides that are complementary to the regions of DNA flanking the segment to be copied. The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately a billion times — that is, one billion copies are made. Such repeated amplification is achieved by the use of a thermostable DNA polymerase, isolated from the bacterium Thermus aquaticus, which remains active during the high-temperature-induced denaturation of double-stranded DNA. The amplified fragment, if desired, can now be used to ligate with a vector for further cloning.
The three steps of every cycle
| Step | What happens | Why the temperature matters |
|---|---|---|
| (i) Denaturation | The double-stranded DNA is separated into two single strands | High temperature breaks the hydrogen bonds between the strands. An ordinary polymerase would be destroyed here; the enzyme from Thermus aquaticus is not |
| (ii) Primer annealing | The two sets of primers bind to their complementary regions flanking the target segment | The temperature is lowered so that base pairing can occur, but kept high enough that only correctly matched primers stay bound |
| (iii) Extension of primers | DNA polymerase extends each primer, using the provided nucleotides and the template strand | Held at the temperature at which the thermostable polymerase works fastest, synthesising the new strand |
Step 4 — Insertion of recombinant DNA into the host
There are several methods of introducing the ligated DNA into recipient cells. Recipient cells, after being made competent to receive it, take up DNA present in their surroundings.
So if a recombinant DNA bearing a gene for resistance to an antibiotic such as ampicillin is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cells. If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, and untransformed recipient cells will die. Because ampicillin resistance allows one to select a transformed cell in the presence of ampicillin, the ampicillin resistance gene in this case is called a selectable marker.
Step 5 — Obtaining the foreign gene product
When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial, plant or animal cell, the alien DNA gets multiplied. But in almost all recombinant technologies the ultimate aim is to produce a desirable protein. Hence there is a need for the recombinant DNA to be expressed, and the foreign gene gets expressed under appropriate conditions. The expression of foreign genes in host cells involves understanding many technical details.
If any protein-encoding gene is expressed in a heterologous host, it is called a recombinant protein. Cells harbouring cloned genes of interest may be grown on a small scale in the laboratory; the cultures may be used for extracting the desired protein and then purifying it using different separation techniques.
Continuous culture
Cells can also be multiplied in a continuous culture system, wherein the used medium is drained out from one side while fresh medium is added from the other, so as to maintain the cells in their physiologically most active log or exponential phase. This type of culturing method produces a larger biomass, leading to higher yields of the desired protein.
Bioreactors
Small-volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors was required, where large volumes — 100 to 1000 litres — of culture can be processed.
A bioreactor can be thought of as a vessel in which raw materials are biologically converted into specific products, individual enzymes and so on, using microbial, plant, animal or human cells. It provides the optimal conditions for achieving the desired product by supplying optimum growth conditions: temperature, pH, substrate, salts, vitamins and oxygen.
The most commonly used bioreactors are of the stirring type. A stirred-tank reactor is usually cylindrical, or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor; alternatively, air can be bubbled through the reactor, giving the sparged stirred-tank type.
| System | Purpose |
|---|---|
| Agitator system | Even mixing of the contents, and distribution of oxygen throughout the vessel |
| Oxygen delivery system | Supplies the oxygen the cells need; in the sparged type, sterile air is bubbled in |
| Foam control system | Controls the foam produced by agitation of a protein-rich medium |
| Temperature control system | Holds the temperature at the optimum for growth and for product formation |
| pH control system | Holds the pH steady as metabolism changes the medium |
| Sampling ports | Allow small volumes of the culture to be withdrawn periodically without opening the vessel |
| Curved base, cylindrical shape | Facilitates mixing of the reactor contents |
📐 Activity: why not just use a bigger flask?
A laboratory shake flask holds 250 ml of culture and produces a usable amount of recombinant protein. A company needs a thousand times as much, and proposes simply to use a thousand-fold bigger shake flask.
Predict: name two things that will go wrong in the giant flask, both of which a stirred-tank bioreactor is designed to prevent.
(i) Oxygen will not reach the middle. A flask is aerated only at its surface, and surface area does not grow in step with volume. The cells in the bulk of the liquid would be starved of oxygen. A bioreactor solves this with an agitator system and an oxygen delivery system — the stirrer facilitates even mixing and oxygen availability throughout the vessel, and air can also be bubbled through.
(ii) Nothing can be controlled or monitored. As the cells grow they change the temperature and pH of the medium, and in a large static vessel these drift out of the optimal range. A bioreactor has a temperature control system, a pH control system and sampling ports, so that small volumes of culture can be withdrawn periodically to check progress without opening — and contaminating — the vessel.
A third, if you want it. Agitating a protein-rich medium produces foam, which is why a bioreactor also has a foam control system. Scale is not just more of the same; above a certain volume, a culture needs engineering.
Step 6 — Downstream processing
After completion of the biosynthetic stage, the product has to be subjected to a series of processes before it is ready for marketing as a finished product. These processes include separation and purification, which are collectively referred to as downstream processing.
The product then has to be formulated with suitable preservatives. Such a formulation has to undergo thorough clinical trials, as in the case of drugs, and strict quality control testing is required for each product. Both the downstream processing and the quality control testing vary from product to product.
Where the real cost lies. The gene, the vector and the transformation are the part of the story that gets told. But for most biotech medicines, the majority of the cost and the time sits after the cells have finished working — in purification, formulation, trials and quality control. Making the molecule is the science; making it safe, stable, and identical in every batch is the engineering.
🎯 Interactive: Walk the Whole Pipeline
Six steps from a cell to a finished product. Choose a step to see what is done, and what you would be left with if you stopped there.
🎯 Competency-Based Questions
Q1. A technician trying to isolate DNA from a fungal culture uses lysozyme, then ribonuclease, then protease, and finally adds chilled ethanol. The yield is almost nil. Identify the error and explain the purpose of each of the other three treatments.
The error. Lysozyme is the wrong enzyme for a fungus. The cells were never opened, so almost no DNA was released, and everything done afterwards operated on an empty solution. Fungi require chitinase, because a fungal wall is made of chitin; lysozyme is for bacteria and cellulase is for plant cells.
What the other three steps are for. Ribonuclease removes the RNA that comes out of the cell along with the DNA. Protease removes proteins — including the histones with which the long DNA molecules are intertwined. Chilled ethanol is what finally precipitates the purified DNA out of solution, where it appears as a collection of fine threads in the suspension.
Why purity matters so much here. In order to cut the DNA with restriction enzymes it needs to be in pure form, free from other macromolecules. Contaminating protein and RNA interfere with digestion and with everything downstream of it.
Q2. PCR would be impossible with the DNA polymerase of a human cell, even though that enzyme copies DNA perfectly well. Explain why, and explain how the difficulty was solved.
Where the difficulty arises. Each PCR cycle begins with denaturation, in which the double-stranded DNA is separated by high temperature. A human DNA polymerase is adapted to about 37°C; at denaturation temperature it is irreversibly denatured itself. You would therefore have to add fresh enzyme at every single cycle, which makes thirty cycles impracticable and hopelessly expensive.
How it was solved. Repeated amplification is achieved by the use of a thermostable DNA polymerase isolated from the bacterium Thermus aquaticus, which remains active during the high-temperature-induced denaturation of double-stranded DNA. Because the enzyme survives every denaturation step, the whole cycle can simply be repeated without adding anything.
The result. If the process of replication is repeated many times, the segment of DNA can be amplified to approximately a billion times. An organism from a hot spring turned out to hold the one component that made automated DNA amplification possible — a good argument for studying organisms that seem to have no use whatsoever.
Q3. Two sets of primers are added to a PCR reaction, not one. Explain what decides which stretch of the genome gets amplified, and what would happen if a single primer were used.
What the primers are. Primers are small, chemically synthesised oligonucleotides that are complementary to the regions of DNA flanking the segment of interest. DNA polymerase extends them using the nucleotides provided in the reaction and the genomic DNA as template.
What decides the product. The primers do, entirely. The enzyme can only start where a primer has annealed and can only synthesise in one direction, so the amplified product is the stretch lying between the two primer sites. Change the two short sequences you put into the tube and the same genomic DNA yields a completely different product — which is why one machine and one enzyme can amplify any gene you like.
With a single primer. Only one strand would be copied and only outward from that one site, with no defined stopping point. There would be no exponential doubling, because each new molecule needs a primer site at both ends for the chain reaction to continue. You would get a small amount of ragged product instead of a billion copies of a defined fragment.
Q4. Besides better aeration and mixing, what other advantages does a stirred-tank bioreactor have over a shake flask? Answer in terms of what the bioreactor can control and observe.
Control of the growth conditions. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions — temperature, pH, substrate, salts, vitamins and oxygen. It has a dedicated temperature control system and pH control system, so these do not simply drift as the cells metabolise. A shake flask offers none of this; it sits at whatever temperature the incubator holds and its pH changes as it pleases.
Observation without contamination. The bioreactor has sampling ports, so small volumes of the culture can be withdrawn periodically to follow the progress of the run. Opening a flask repeatedly risks losing the whole batch to contamination.
Foam control. Agitation of a protein-rich medium generates foam, which can carry cells out of the liquid and block filters; the bioreactor includes a foam control system.
Scale, and therefore economics. Small-volume cultures cannot yield appreciable quantities of products, whereas a bioreactor processes 100 to 1000 litres of culture. It can also be run as a continuous culture system, with used medium drained from one side while fresh medium is added from the other, keeping the cells in their physiologically most active log phase and so producing a larger biomass and higher yields.
Q5. A company has 900 litres of culture in which its engineered cells have expressed the target protein successfully. Explain why the product is still many months away from a pharmacy shelf.
Because the biosynthetic stage is only the first half. After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product. These processes — separation and purification — are collectively referred to as downstream processing.
What remains to be done. First, the protein must be separated from a thousand litres of cells, medium and cellular debris, and purified to a defined standard. Then the product has to be formulated with suitable preservatives, so that it is stable and deliverable. That formulation has to undergo thorough clinical trials, as in the case of drugs. And strict quality control testing is required for each product, on every batch.
Why none of this can be standardised away. The downstream processing and quality control testing vary from product to product. A protein that is stable in solution and one that must be freeze-dried, a product injected into the bloodstream and one taken by mouth, present entirely different purification and formulation problems.
The point to take away. Expression proves the biology works. Downstream processing is what turns a successful experiment into a medicine.
🧠 Assertion–Reason Questions
For each pair, decide whether both statements are true and whether the reason correctly explains the assertion.
Assertion (A): PCR uses a DNA polymerase isolated from Thermus aquaticus.
Reason (R): This enzyme remains active during the high-temperature-induced denaturation of double-stranded DNA.
Both A and R are true, and R is the correct explanation of A.
Repeated amplification is achieved precisely because the enzyme survives the denaturation step of every cycle. A heat-labile polymerase would have to be replaced each round, which would make thirty cycles impossible in practice.
Assertion (A): A continuous culture system gives higher yields of the desired protein than a batch culture.
Reason (R): Used medium is drained out from one side while fresh medium is added from the other, maintaining the cells in their physiologically most active log phase.
Both A and R are true, and R is the correct explanation of A.
This type of culturing method produces a larger biomass, leading to higher yields of the desired protein. In a batch culture the cells inevitably exhaust the medium and leave the log phase; continuous culture prevents that from happening.
Assertion (A): Downstream processing is the same for every biotechnological product.
Reason (R): Downstream processing consists of separation and purification.
A is false but R is true.
Downstream processing does indeed consist of separation and purification, followed by formulation with suitable preservatives, clinical trials where applicable and strict quality control testing. But it is explicitly not the same for every product: the downstream processing and quality control testing vary from product to product.
Frequently Asked Questions - Processes of Recombinant DNA Technology
What are the steps of recombinant DNA technology in order?
Isolation of DNA, fragmentation of DNA by restriction endonucleases, isolation of a desired DNA fragment, ligation of the DNA fragment into a vector, transferring the recombinant DNA into the host, culturing the host cells in a medium at large scale, and extraction of the desired product.
Which enzymes are used to break open cells during DNA isolation?
Lysozyme is used for bacteria, cellulase for plant cells and chitinase for fungus. Each digests the material of that group's cell wall so that the DNA can be released along with other macromolecules.
How is DNA purified after the cell has been broken open?
RNA is removed by treatment with ribonuclease and proteins are removed by treatment with protease, since genes lie on long DNA molecules intertwined with proteins such as histones. Other molecules are removed by appropriate treatments, and the purified DNA finally precipitates out after the addition of chilled ethanol, appearing as a collection of fine threads in the suspension.
What is PCR and what are the three steps of each cycle?
PCR stands for Polymerase Chain Reaction, in which multiple copies of the gene or DNA of interest are synthesised in vitro using two sets of primers and the enzyme DNA polymerase. Each cycle has three steps: denaturation, primer annealing, and extension of primers.
What are primers in PCR?
Primers are small, chemically synthesised oligonucleotides that are complementary to the regions of DNA flanking the segment to be amplified. DNA polymerase extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
Why is a thermostable DNA polymerase needed for PCR?
Because each cycle begins with high-temperature denaturation of double-stranded DNA, which would destroy an ordinary polymerase. The thermostable DNA polymerase isolated from the bacterium Thermus aquaticus remains active during this high temperature step, so the cycle can be repeated many times and the segment of DNA can be amplified to approximately a billion times.
What is a recombinant protein?
If any protein encoding gene is expressed in a heterologous host, that is a host other than the organism the gene came from, it is called a recombinant protein.
What is a continuous culture system and why is it used?
In a continuous culture system the used medium is drained out from one side while fresh medium is added from the other, so as to maintain the cells in their physiologically most active log or exponential phase. This type of culturing method produces a larger biomass, leading to higher yields of the desired protein.
What is a bioreactor and what systems does it contain?
A bioreactor is a vessel in which raw materials are biologically converted into specific products, individual enzymes and so on, using microbial, plant, animal or human cells, and in which large volumes of 100 to 1000 litres of culture can be processed. It provides optimum growth conditions of temperature, pH, substrate, salts, vitamins and oxygen. The most commonly used bioreactors are of the stirring type, and a bioreactor has an agitator system, an oxygen delivery system, a foam control system, a temperature control system, a pH control system and sampling ports.
What is downstream processing?
After the biosynthetic stage is complete, the product is put through a series of processes before it is ready for marketing as a finished product. These include separation and purification, which are collectively referred to as downstream processing. The product then has to be formulated with suitable preservatives, the formulation has to undergo thorough clinical trials as in the case of drugs, and strict quality control testing is required for each product. Downstream processing and quality control testing vary from product to product.
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