Recombinant DNA Technology

BiologyBiotechnology: Principles and ProcessesFor NEET aspirants

Recombinant DNA technology lets scientists take a desired gene from one organism, join it to a vector and multiply it inside a host cell to obtain a useful product. This page explains what biotechnology means, its two core techniques, the first recombinant DNA made by Cohen and Boyer, and every step of the process, from isolating DNA to PCR, bioreactors and downstream processing, as in the NCERT Class 12 chapter Biotechnology: Principles and Processes. NEET often tests the order of these steps, so learn recombinant DNA technology step by step.

On this page1Biotechnology2Principles3First recombinant DNA4Steps in order5Isolating DNA6Cutting and joining7PCR8Host and product9Bioreactors10Downstream processing11Exam essentials12Quick revision13Solved examples14Practice

Key Points at a Glance

★ Must learn Biotechnology: using live organisms, or enzymes from organisms, to make products and processes useful to humans.
EFB definition: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.
★ Must learn Two core techniques: genetic engineering (alters DNA or RNA and changes the host's phenotype) and bioprocess engineering (sterile, large-scale growth of the desired cell).
An alien piece of DNA multiplies in a host only when it is linked to an origin of replication (ori).
★ Must learn First recombinant DNA: Stanley Cohen and Herbert Boyer, 1972; an antibiotic resistance gene joined to a native plasmid of Salmonella typhimurium, then cloned in Escherichia coli.
Three basic steps: identify the DNA with desirable genes, introduce it into the host, maintain it in the host and pass it to the progeny.
★ Must learn Order of the process: isolate DNA, cut it, isolate the desired fragment, ligate it into a vector, transfer it into the host, culture on a large scale, extract the product.
Cells are opened with lysozyme (bacteria), cellulase (plant cells) or chitinase (fungus); purified DNA precipitates with chilled ethanol.
★ Must learn PCR: two sets of primers and a thermostable DNA polymerase from Thermus aquaticus; denaturation, primer annealing, extension; about 1 billion copies.
Recombinant protein: the protein made when a protein-encoding gene is expressed in a heterologous host.
Bioreactors process 100-1000 litres of culture; the stirred-tank type is the most common.
Downstream processing: separation and purification, then formulation, clinical trials and quality control.

1. What is Biotechnology?

1.1 How biotechnology arose

Since the time of René Descartes, the seventeenth-century French philosopher, mathematician and biologist, human knowledge has aimed at technologies that add comfort and value to human life.
This made the study of nature anthropocentric: centred on human needs.
Physics and chemistry gave rise to engineering, technologies and industries for human comfort and welfare.
The major use of the biological world is as a source of food.
Biotechnology is the twentieth-century off-shoot of modern biology. Its products brought a qualitative improvement in health and food production.

1.2 Meaning of biotechnology

★ Very important Biotechnology: the techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.

In this broad sense, making curd, bread or wine is also biotechnology, because all three are microbe-mediated processes.
★ Exam imp Today the term is used in a restricted sense: processes that use genetically modified organisms (GMOs) to do the same on a larger scale.
A genetically modified organism is one whose genetic material has been altered by genetic engineering.
Many other techniques also count as biotechnology:

Technique	What it does
In vitro fertilisation	Leads to a 'test-tube' baby
Synthesising a gene	A gene is made and then used
DNA vaccine	A vaccine is developed from DNA
Gene correction	A defective gene is corrected

★ Exam imp The European Federation of Biotechnology (EFB) gives a definition that covers both the traditional view and modern molecular biotechnology: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Memory Trick Science meets life for products and services: the EFB definition joins natural science with four biological inputs (organisms, cells, parts thereof, molecular analogues) and gives two outputs (products and services).

Key idea

Curd, bread and wine fit the broad meaning; modern biotechnology uses genetically modified organisms on a large scale.

2. Principles of Biotechnology

2.1 The two core techniques

Among many techniques, two core techniques enabled the birth of modern biotechnology.

Genetic engineering

Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce it into host organisms and thus change the phenotype of the host.

Bioprocess engineering

Maintaining a sterile (microbial contamination-free) ambience in chemical engineering processes, so that only the desired microbe or eukaryotic cell grows in large quantities. It makes products like antibiotics, vaccines and enzymes.

Memory Trick Change the gene, then grow it clean: genetic engineering changes the gene; bioprocess engineering grows the changed cells in bulk without contamination.

2.2 Why genetic engineering?

Sexual reproduction gives variations and unique combinations of genes; some help the organism and the population.
Asexual reproduction preserves the genetic information, while sexual reproduction permits variation.
★ Exam imp Traditional hybridisation in plant and animal breeding often brings in and multiplies undesirable genes along with the desired genes.
Genetic engineering overcomes this limitation. Its techniques include the creation of recombinant DNA, gene cloning and gene transfer.
It isolates and introduces only one or a set of desirable genes into the target organism, without the undesirable genes.

Traditional hybridisation

Whole genomes are mixed, so undesirable genes come in and multiply along with the desired ones.

Genetic engineering

Only the chosen gene or set of genes is moved, so no undesirable genes are introduced.

2.3 Origin of replication and cloning

A piece of DNA transferred into an alien organism usually cannot multiply in the progeny cells.
If it gets integrated into the genome of the recipient, it may multiply and be inherited along with the host DNA.
This happens because the alien DNA has become part of a chromosome, which can replicate.

Origin of replication (ori): a specific DNA sequence in a chromosome that is responsible for initiating replication. An alien DNA is linked to an ori so that it can replicate and multiply in the host.

★ Exam imp Cloning: making multiple identical copies of any template DNA. Multiplying an alien DNA linked to an ori is also called cloning.

Key idea

Genetic engineering moves only the desired genes, and an ori lets the moved DNA multiply in the host.

3. The First Recombinant DNA

3.1 Herbert Boyer and Stanley Cohen

Herbert Boyer was born in 1936 and grew up in western Pennsylvania, a region of railroads and mines.
He completed graduate work at the University of Pittsburgh in 1963, then spent three years in post-graduate studies at Yale.
In 1966 he became an assistant professor at the University of California at San Francisco.
★ Exam imp By 1969 he had studied restriction enzymes of the E. coli bacterium. These enzymes cut DNA strands in a way that left 'sticky ends'.
The sticky ends made pasting together pieces of DNA a precise exercise.
This led to a conversation in Hawaii with Stanley Cohen, a scientist at Stanford.
Cohen studied plasmids: small rings of DNA that float freely in the cytoplasm of certain bacteria and replicate independently of the chromosome.
Cohen had developed a method of removing plasmids from a cell and reinserting them in other cells.
Combining plasmid transfer with DNA splicing, Boyer and Cohen recombined DNA segments in desired forms and inserted them into bacteria.
These bacteria then acted as manufacturing plants for specific proteins. This breakthrough founded the discipline of biotechnology.

3.2 How the first recombinant DNA was made

★ Exam imp The first recombinant DNA linked a gene encoding antibiotic resistance with a native plasmid of Salmonella typhimurium.
Plasmid: an autonomously replicating, circular, extra-chromosomal DNA.
Stanley Cohen and Herbert Boyer achieved this in 1972.

Cut: the antibiotic resistance gene was isolated by cutting out a piece of DNA from a plasmid that conferred antibiotic resistance. Restriction enzymes, the 'molecular scissors', made cutting at specific locations possible.
Join: the cut piece of DNA was linked with the plasmid DNA, which acts as a vector. The linking was done by the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. This gave a new circular, autonomously replicating DNA made in vitro: recombinant DNA.
Transfer: the recombinant DNA was transferred into Escherichia coli, a bacterium closely related to Salmonella.
Copy: it replicated using the new host's DNA polymerase and made multiple copies. This was called cloning of the antibiotic resistance gene in E. coli.

★ Very important Recombinant DNA: DNA composed of DNA from different sources or genomes, joined by DNA ligase. In the first experiment it was a new combination of circular, autonomously replicating DNA created in vitro.

Vector: a carrier. A mosquito is an insect vector that carries the malarial parasite into the human body; in the same way, a plasmid carries an alien piece of DNA into the host.

Memory Trick Cut, Join, Transfer, Copy: the four moves of the 1972 experiment. The donor of the plasmid was Salmonella; the host that copied it was E. coli.

3.3 Three basic steps in genetically modifying an organism

Identification of DNA with desirable genes.
Introduction of the identified DNA into the host.
Maintenance of the introduced DNA in the host and its transfer to the progeny.

Memory Trick Identify, Introduce, Inherit: find the gene, put it in, and make sure the progeny inherits it.

Key idea

Cohen and Boyer (1972) cut, joined and cloned an antibiotic resistance gene: the model for every later gene transfer.

4. Processes of Recombinant DNA Technology: Steps in Order

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 DNA fragment into a vector.
Transferring the recombinant DNA into the host.
Culturing the host cells in a medium at a large scale.
Extraction of the desired product.

Memory Trick I Can Isolate Lots, Then Culture Everything: Isolate DNA, Cut it, Isolate the fragment, Ligate, Transfer, Culture, Extract.

NEET Focus

Sequence questions on this process are common. Two checks clear most traps: ligation comes before transfer into the host, and large-scale culture comes before extraction of the product. PCR, when used, amplifies the gene of interest before it is ligated into a vector.

5. Isolation of the Genetic Material (DNA)

Nucleic acid is the genetic material of all organisms without exception; in the majority of organisms it is deoxyribonucleic acid (DNA).
To be cut by restriction enzymes, DNA must be in pure form, free from other macromolecules.
DNA is enclosed within membranes, so the cell is broken open. This releases DNA along with RNA, proteins, polysaccharides and lipids.
Genes lie on long DNA molecules intertwined with proteins such as histones.

Treat the bacterial cells, or the plant or animal tissue, with enzymes to break the cells open: lysozyme for bacteria, cellulase for plant cells, chitinase for fungus.
Remove RNA with ribonuclease.
Remove proteins with protease.
Remove other molecules by appropriate treatments.
Add chilled ethanol: the purified DNA precipitates out as a collection of fine threads in the suspension.
Remove the DNA by spooling: winding the threads onto a glass rod.

Enzyme or reagent	Acts on	Role in isolating DNA
Lysozyme	Bacterial cells	Breaks the cell open
Cellulase	Plant cells	Breaks the cell open
Chitinase	Fungus	Breaks the cell open
Ribonuclease	RNA	Removes RNA
Protease	Proteins (such as histones)	Removes proteins
Chilled ethanol	Purified DNA	Makes DNA precipitate as threads

Figure 1: DNA that separates out can be removed by spooling. Left: after chilled ethanol is added, purified DNA appears as fine threads in the suspension. Right: the threads are wound onto a glass rod and lifted out.

Memory Trick Name the wall, name the enzyme: the bacterial wall is broken by lysozyme; the plant wall is made of cellulose, so cellulase; the fungal wall is made of chitin, so chitinase.

Tips and Tricks Read the enzyme's name: the stem tells you the target. Cellul-ase acts on cellulose, chitin-ase on chitin, ribonucle-ase on RNA (ribonucleic acid) and prote-ase on proteins. Ethanol is not an enzyme: it only precipitates DNA.

Quick Recall: tap to check

Which enzyme opens a fungal cell to release DNA?

Chitinase.

How are proteins such as histones removed from the DNA preparation?

By treatment with protease.

What makes the purified DNA precipitate, and how does it appear?

Chilled ethanol; the DNA appears as a collection of fine threads in the suspension.

Key idea

Open the cell, digest away RNA and proteins, then precipitate pure DNA with chilled ethanol and spool it out.

6. Cutting of DNA at Specific Locations

Restriction enzyme digestion: purified DNA is incubated with the restriction enzyme at the optimal conditions for that specific enzyme.
★ Exam imp Agarose gel electrophoresis is used to check the progress of a restriction enzyme digestion.
DNA is a negatively charged molecule, so it moves towards the positive electrode (anode) in the gel.
The process is repeated with the vector DNA also.

6.1 Joining the gene of interest to the vector

Cut the source DNA and the vector DNA with the same specific restriction enzyme.
Mix the cut-out 'gene of interest' with the cut vector that now has a space.
Add ligase: the gene joins the vector, giving recombinant DNA.

The tools used here, restriction enzymes, gel electrophoresis and vectors, are covered in detail under Tools of Recombinant DNA Technology.

Key idea

Cut source and vector with the same enzyme, check the cut on a gel, then join them with ligase.

7. Amplification of Gene of Interest using PCR

★ Very important PCR (Polymerase Chain Reaction): a 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.

Primers: small, chemically synthesised oligonucleotides that are complementary to regions of the DNA.
The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
★ Exam imp When replication is repeated many times, the DNA segment is amplified about a billion times: about 1 billion copies are made.
This repeated amplification uses a thermostable DNA polymerase isolated from the bacterium Thermus aquaticus (Taq polymerase).
It stays active during the high temperature used to denature double-stranded DNA.
The amplified fragment can then be ligated with a vector for further cloning.

7.1 The PCR cycle

Denaturation: heat separates the two strands of the double-stranded (ds) DNA.
Primer annealing: the two primers bind to their complementary regions at the two ends of the region to be amplified.
Extension of primers: DNA polymerase (Taq polymerase) with deoxynucleotides extends each primer along its template.
The cycle is repeated (about 30 cycles); the region is amplified about 1 billion times.

Test yourself: hide labels

Figure 2: Polymerase chain reaction (PCR). Each cycle has three steps: denaturation, primer annealing and extension of primers. About 30 cycles amplify the region about a billion times.

Memory Trick Heat, Hook, Build: heat separates the strands (denaturation), primers hook onto the template (annealing), polymerase builds the new strand (extension).

Memory Trick Taq = Thermus aquaticus: 'T' for Thermus and 'aq' for aquaticus. Thermus also reminds you that the enzyme is thermostable.

Extra Depth: Each PCR cycle doubles the target DNA, so one molecule gives

2^{n}

copies after

n

cycles. After 30 cycles,

2^{30} \approx 1.07 \times 1 0^{9}

, which is the 'about 1 billion' copies.

NEET Focus

PCR facts that are asked together: it works in vitro; it needs two sets of primers, deoxynucleotides, a template and DNA polymerase; the polymerase is thermostable Taq polymerase from Thermus aquaticus; the order is denaturation, annealing, extension. A statement that the polymerase is destroyed at the denaturation temperature is false.

Quick Recall: tap to check

Name the three steps of each PCR cycle, in order.

Denaturation, primer annealing and extension of primers.

Why must the DNA polymerase used in PCR be thermostable?

It must stay active during the high temperature that denatures the double-stranded DNA in every cycle.

About how many copies of the DNA segment does repeated PCR produce?

About 1 billion copies (amplified about a billion times).

Key idea

PCR copies a chosen DNA segment in vitro about a billion times, using primers and heat-stable Taq polymerase.

8. Insertion of Recombinant DNA into the Host Cell or Organism

There are several methods of introducing the ligated DNA into recipient cells.
Recipient cells are first made 'competent': able to take up DNA present in their surroundings.
Suppose a recombinant DNA carrying a gene for resistance to an antibiotic (for example, ampicillin) is transferred into E. coli cells.
The host cells become transformed into ampicillin-resistant cells.
★ Exam imp When the cells are spread on agar plates containing ampicillin, only the transformants grow; untransformed recipient cells die.
The ampicillin resistance gene lets us select transformed cells, so it is called a selectable marker.

Selectable marker: a gene, such as the ampicillin resistance gene, that allows only transformed cells to grow on a selective medium, so that they can be picked out.

Key idea

Competent cells take up recombinant DNA, and an antibiotic plate lets only the transformed cells survive.

9. Obtaining the Foreign Gene Product

When alien DNA is inserted into a cloning vector and transferred into a bacterial, plant or animal cell, the alien DNA gets multiplied.
★ Exam imp In almost all recombinant technologies, the ultimate aim is to produce a desirable protein.
So the recombinant DNA must be expressed. The foreign gene is expressed under appropriate conditions; this involves many technical details.
After the gene is cloned and the conditions to induce expression of the target protein are optimised, the protein is produced on a large scale.

★ Exam imp Recombinant protein: the protein formed when any protein-encoding gene is expressed in a heterologous host, that is, a host of a different species.

9.1 Small-scale and continuous culture

Cells harbouring the cloned gene may be grown on a small scale in the laboratory.
These cultures are used to extract the desired protein and purify it by different separation techniques.
Cells can also be multiplied in a continuous culture system.

Used medium is drained out from one side.
Fresh medium is added from the other side.
The cells stay in their physiologically most active log (exponential) phase.
This gives a larger biomass and higher yields of the desired protein.

9.2 Bioreactors

Small-volume cultures cannot yield appreciable quantities of products.
★ Exam imp Large-scale production needed bioreactors, in which large volumes (100-1000 litres) of culture can be processed.

Bioreactors: vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial, plant, animal or human cells.

A bioreactor gives the optimal conditions for the desired product by providing 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 has a curved base, which helps mix the reactor contents.
The stirrer gives even mixing and oxygen availability throughout the bioreactor.
Alternatively, air can be bubbled through the reactor (sparging).

Test yourself: hide labels

Figure 3: Stirred-tank bioreactors. (a) A simple stirred-tank bioreactor; (b) a sparged stirred-tank bioreactor, through which sterile air bubbles are sparged. The bubbles greatly increase the area for oxygen transfer.

System of a stirred-tank bioreactor	Part or role
Agitator system	Motor turning the flat bladed impeller (stirrer)
Oxygen delivery system	Sterile air entering at the base; sparged air bubbles
Foam control system	Foam breaker
Temperature control system	Keeps the culture broth at its optimum temperature
pH control system	Inlet for acid or base
Sampling ports	Small volumes of culture are withdrawn periodically

The vessel is sterilised with steam (steam for sterilisation).

Simple stirred-tank

A motor-driven flat bladed impeller mixes the culture broth; sterile air enters at the base.

Sparged stirred-tank

Sterile air is sparged as bubbles. The bubbles dramatically increase the oxygen transfer area; the surface area for oxygen transfer increases and gas is entrained.

Memory Trick Stir, Air, Foam; Heat, Acid, Sample: agitator, oxygen delivery, foam control, temperature control, pH control and sampling ports, the six systems of a stirred-tank bioreactor.

Memory Trick Two settings and four supplies: a bioreactor sets temperature and pH, and supplies substrate, salts, vitamins and oxygen.

Quick Recall: tap to check

What volume of culture can a bioreactor process?

Large volumes, 100-1000 litres.

Why does a continuous culture give a higher yield?

Draining used medium and adding fresh medium keeps the cells in the log (exponential) phase, giving a larger biomass.

Which part of a bioreactor controls foam?

The foam breaker (foam control system).

Key idea

The aim is a protein: express the gene, then grow the cells in a stirred-tank bioreactor of 100-1000 litres under optimum conditions.

10. Downstream Processing

After the biosynthetic stage is complete, the product goes through a series of processes before it is ready for marketing as a finished product.

★ Very important Downstream processing: the processes of separation and purification of the product after the biosynthetic stage.

Separation of the product.
Purification of the product.
Formulation with suitable preservatives.
Thorough clinical trials of the formulation, as in the case of drugs.
Strict quality control testing of each product.

Downstream processing and quality control testing vary from product to product.

Memory Trick Separate, Purify, Preserve, Trial, Test: the route from culture broth to a marketed product.

Key idea

Downstream processing turns the crude product into a safe, pure, marketable formulation.

11. Exam Essentials

Pairs to Match

Item	Matches with
Stanley Cohen and Herbert Boyer	First recombinant DNA (1972)
Herbert Boyer	Restriction enzymes of E. coli leaving sticky ends (1969)
Stanley Cohen	Plasmids; method to remove and reinsert them
European Federation of Biotechnology (EFB)	Definition covering traditional and modern biotechnology
Genetic engineering	Alters DNA or RNA; changes the host's phenotype
Bioprocess engineering	Sterile, large-scale growth of the desired cells
Origin of replication (ori)	Initiates replication of the linked DNA
Salmonella typhimurium	Native plasmid used in the first recombinant DNA
Escherichia coli	Host in which the first recombinant DNA was cloned
DNA ligase	Joins the ends of cut DNA molecules
Lysozyme / cellulase / chitinase	Bacteria / plant cells / fungus
Chilled ethanol	Precipitates purified DNA
Thermus aquaticus	Thermostable DNA polymerase for PCR
Continuous culture	Cells kept in the log (exponential) phase
Downstream processing	Separation and purification of the product

Exceptions

An alien DNA does not multiply in a host unless it is integrated into a chromosome or linked to an ori.
Curd, bread and wine count as biotechnology only in the broad sense; the modern, restricted sense needs genetically modified organisms.
Traditional hybridisation does not keep out undesirable genes; genetic engineering does.
Taq polymerase is not inactivated at the high temperature of denaturation.
On an ampicillin plate, untransformed cells die; only transformants grow.
Small-volume cultures cannot give appreciable quantities of product.
E. coli was the host of the first recombinant DNA, not the source of its plasmid.

Numbers to Remember

Seventeenth century: René Descartes.
1936: Boyer born; 1963: University of Pittsburgh; 1966: University of California at San Francisco; 1969: work on restriction enzymes.
1972: first recombinant DNA by Cohen and Boyer.
2 core techniques of modern biotechnology; 3 basic steps in genetically modifying an organism.
3 steps per PCR cycle; about 30 cycles; about 1 billion copies.
100-1000 litres: culture volume handled by bioreactors.

Examples to Remember

Type	Examples on this page
Microbe-mediated (traditional) processes	Curd, bread, wine
Other biotechnological techniques	In vitro fertilisation (test-tube baby), gene synthesis, DNA vaccine, correcting a defective gene
Products of bioprocess engineering	Antibiotics, vaccines, enzymes
Cell-opening enzymes	Lysozyme, cellulase, chitinase
Organisms in the first recombinant DNA	Salmonella typhimurium (plasmid), Escherichia coli (host)
Selectable marker antibiotic	Ampicillin

12. Quick Revision

Biotechnology uses live organisms or their enzymes to make useful products and processes.
Modern (restricted) biotechnology uses genetically modified organisms on a large scale.
EFB: integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services.
Core techniques: genetic engineering and bioprocess engineering.
Genetic engineering moves only desired genes; hybridisation also carries undesirable ones.
Alien DNA multiplies only when linked to an origin of replication; this is cloning.
1972: Cohen and Boyer joined an antibiotic resistance gene to a Salmonella typhimurium plasmid and cloned it in E. coli.
Three steps: identify the DNA, introduce it, maintain it and pass it to the progeny.
Process: isolate DNA, cut, isolate fragment, ligate, transfer, culture, extract.
Lysozyme, cellulase and chitinase open cells; ribonuclease and protease clean the DNA; chilled ethanol precipitates it.
Gel electrophoresis checks digestion; DNA moves to the anode.
PCR: primers, Taq polymerase, denaturation, annealing, extension; about a billion copies.
Ampicillin plates select transformants; the resistance gene is a selectable marker.
Recombinant protein: a gene expressed in a heterologous host; continuous culture keeps cells in log phase.
In short: restriction endonucleases, DNA ligase and plasmid or viral vectors isolate and ferry foreign DNA into a host, where it is expressed and its protein purified.
Bioreactors (100-1000 L, stirred-tank) then downstream processing: separation, purification, formulation, trials, quality control.

13. Solved Examples

Solved Example 1

Match List I with List II.
List I: A. Lysozyme, B. Cellulase, C. Chitinase, D. Ribonuclease
List II: I. Fungal cells, II. Removal of RNA, III. Bacterial cells, IV. Plant cells
Choose the correct answer:
(A) A-III, B-IV, C-I, D-II
(B) A-IV, B-III, C-I, D-II
(C) A-III, B-I, C-IV, D-II
(D) A-II, B-IV, C-I, D-III

Solution:

Answer: (A). Lysozyme opens bacterial cells (III), cellulase plant cells (IV), chitinase fungal cells (I), and ribonuclease removes RNA (II).

Solved Example 2

Read the statements.
A. PCR synthesises multiple copies of a gene in vitro.
B. The thermostable DNA polymerase used in PCR comes from Thermus aquaticus.
C. Each PCR cycle has denaturation, primer annealing and extension.
D. The polymerase is inactivated during denaturation, so fresh enzyme is added every cycle.
E. Repeated amplification can give about 1 billion copies.
Choose the correct answer:
(A) A, B and C only
(B) A, B, C and E only
(C) B, C and D only
(D) A, D and E only

Solution:

Answer: (B). D is wrong: Taq polymerase is thermostable and stays active during the high temperature of denaturation. The other four statements are correct.

Solved Example 3

Arrange these steps of recombinant DNA technology in the correct order.
A. Ligation of the DNA fragment into a vector
B. Isolation of DNA
C. Transferring the recombinant DNA into the host
D. Fragmentation of DNA by restriction endonucleases
E. Culturing the host cells at a large scale
Choose the correct answer:
(A) B, D, A, C, E
(B) B, A, D, C, E
(C) D, B, A, E, C
(D) B, D, C, A, E

Solution:

Answer: (A). DNA is isolated (B) and cut (D), then ligated into the vector (A), transferred into the host (C) and finally cultured at a large scale (E).

Solved Example 4

Statement I: Bioprocess engineering keeps a sterile ambience so that only the desired microbe or eukaryotic cell grows.
Statement II: Traditional hybridisation introduces only the desired genes into the target organism.
Choose the correct answer:
(A) Both Statement I and Statement II are correct
(B) Both Statement I and Statement II are incorrect
(C) Statement I is correct but Statement II is incorrect
(D) Statement I is incorrect but Statement II is correct

Solution:

Answer: (C). Statement I is the definition of bioprocess engineering. Statement II is wrong: hybridisation often brings in undesirable genes too; only genetic engineering avoids them.

Solved Example 5

Which of the following is NOT a system of a stirred-tank bioreactor?
(A) Foam control system
(B) pH control system
(C) Sampling ports
(D) Ethidium bromide staining system

Solution:

Answer: (D). A stirred-tank bioreactor has agitator, oxygen delivery, foam control, temperature control and pH control systems and sampling ports. Ethidium bromide is used to stain DNA in gels.

Solved Example 6

In the first recombinant DNA, the antibiotic resistance gene was linked with a native plasmid of:
(A) Escherichia coli
(B) Salmonella typhimurium
(C) Thermus aquaticus
(D) Agrobacterium tumefaciens

Solution:

Answer: (B). The plasmid was native to Salmonella typhimurium; E. coli was the host in which the recombinant DNA was cloned.

14. Practice Questions

Practice Questions

List 10 recombinant proteins used in medical practice and state where each is used as a therapeutic.Answer: Human insulin (diabetes), human growth hormone (growth hormone deficiency), interferon alpha (hepatitis and some cancers), interferon beta (multiple sclerosis), erythropoietin (anaemia of kidney failure), tissue plasminogen activator (dissolving clots in heart attack and stroke), clotting factor VIII (haemophilia A), hepatitis B vaccine (surface antigen, for vaccination), granulocyte colony-stimulating factor (low white cell counts after chemotherapy) and interleukin-2 (some cancers).
What would be the molar concentration of human DNA in a human cell?Answer: A diploid human cell has 46 DNA molecules, one per chromosome: $46 \div (6.022 \times 1 0^{23}) \approx 7.6 \times 1 0^{- 23}$ mol. The DNA lies in the nucleus. Taking the nucleus as a sphere 10 micrometres across (volume about $5.2 \times 1 0^{- 13}$ L), the concentration in the nucleus is about $1.5 \times 1 0^{- 10}$ mol per litre; a nucleus 6 micrometres across gives about $7 \times 1 0^{- 10}$ mol per litre. The value depends on the volume assumed.
Besides better aeration and mixing, what other advantages do stirred-tank bioreactors have over shake flasks?Answer: They process large volumes (100-1000 L); they control temperature, pH and foam; they supply oxygen evenly; they have sampling ports for withdrawing small volumes; and they keep a sterile environment, so yields are far higher.
Recall meiosis and indicate at what stage a recombinant DNA is made.Answer: Natural recombinant DNA forms by crossing over between non-sister chromatids of homologous chromosomes during pachytene of prophase I.
Describe briefly: (a) bioreactors; (b) downstream processing.Answer: (a) Vessels of 100-1000 L in which raw materials are converted into products by cells under optimum temperature, pH, substrate, salts, vitamins and oxygen; usually stirred tanks. (b) Separation and purification of the product after the biosynthetic stage, then formulation with preservatives, clinical trials and quality control.
Explain briefly: (a) PCR; (b) chitinase.Answer: (a) In vitro amplification of a gene using two sets of primers and thermostable Taq polymerase, in cycles of denaturation, annealing and extension, giving about a billion copies. (b) An enzyme that breaks the chitin wall of fungal cells to release DNA.
Distinguish between RNA and DNA.Answer: RNA has ribose sugar and uracil, and is usually single-stranded; DNA has deoxyribose and thymine, and is double-stranded. DNA is the genetic material of most organisms; RNA is removed with ribonuclease during DNA isolation.
Match List I with List II.
List I: A. Stanley Cohen and Herbert Boyer, B. Thermus aquaticus, C. European Federation of Biotechnology, D. Continuous culture
List II: I. Cells kept in log phase, II. Definition of biotechnology, III. Thermostable DNA polymerase, IV. First recombinant DNA
Choose the correct answer:
(A) A-IV, B-III, C-II, D-I
(B) A-III, B-IV, C-II, D-I
(C) A-IV, B-II, C-III, D-I
(D) A-IV, B-III, C-I, D-IIAnswer: (A). Cohen and Boyer made the first recombinant DNA; Thermus aquaticus gives Taq polymerase; EFB defined biotechnology; continuous culture keeps cells in log phase.
Read the statements.
A. Lysozyme is used to break open bacterial cells.
B. Purified DNA precipitates on adding chilled ethanol.
C. RNA is removed by treatment with protease.
D. Genes lie on DNA intertwined with proteins such as histones.
Choose the correct answer:
(A) A, B and C only
(B) A, B and D only
(C) B, C and D only
(D) A, C and D onlyAnswer: (B). C is wrong: RNA is removed by ribonuclease; protease removes proteins.
Arrange the events of the 1972 experiment in order.
A. DNA ligase joins the gene to the plasmid
B. The antibiotic resistance gene is cut out of a plasmid
C. The recombinant DNA is transferred into E. coli
D. The DNA is copied using the host's DNA polymerase
(A) B, A, C, D
(B) A, B, C, D
(C) B, C, A, D
(D) C, B, A, DAnswer: (A). Cut the gene, join it with ligase, transfer it into the host, then it is copied.
Statement I: A continuous culture system keeps cells in their log (exponential) phase.
Statement II: Small-volume laboratory cultures yield appreciable quantities of product.
Choose the correct answer:
(A) Both Statement I and Statement II are correct
(B) Both Statement I and Statement II are incorrect
(C) Statement I is correct but Statement II is incorrect
(D) Statement I is incorrect but Statement II is correctAnswer: (C). Statement II is wrong: small cultures cannot give appreciable quantities, which is why bioreactors are needed.
Which of the following is NOT one of the three basic steps in genetically modifying an organism?
(A) Identification of DNA with desirable genes
(B) Introduction of the identified DNA into the host
(C) Maintenance of the DNA in the host and transfer to its progeny
(D) Downstream processing of the productAnswer: (D). Downstream processing comes after production; it is not one of the three basic steps.

Common Mistakes to Avoid

Watch out

Taking E. coli as the source of the first plasmid. Correct: the plasmid was native to Salmonella typhimurium; E. coli was the host.
Crediting the first recombinant DNA to the wrong scientists. Correct: Stanley Cohen and Herbert Boyer, 1972.
Using cellulase to open fungal cells. Correct: chitinase for fungus, cellulase for plant cells, lysozyme for bacteria.
Removing RNA with protease. Correct: ribonuclease removes RNA; protease removes proteins.
Saying Taq polymerase comes from E. coli. Correct: from Thermus aquaticus; it is thermostable.
Placing ligation after transfer into the host. Correct: the fragment is ligated into the vector first, then transferred.
Defining downstream processing as growing the cells. Correct: it is separation and purification after the biosynthetic stage.
Calling curd-making modern biotechnology. Correct: it fits only the broad sense; the restricted sense needs genetically modified organisms.

Frequently Asked Questions

What is recombinant DNA technology?

Recombinant DNA technology, or genetic engineering, joins DNA from different sources to make recombinant DNA. A gene of interest is cut with a restriction enzyme, ligated into a vector such as a plasmid, transferred into a host, multiplied, and expressed to give a useful protein, which is then purified by downstream processing.

What are the two core techniques of modern biotechnology?

Genetic engineering alters the chemistry of DNA or RNA, introduces it into a host and changes the host's phenotype. Bioprocess engineering keeps a sterile environment so that only the desired microbe or eukaryotic cell grows in large quantities, to make antibiotics, vaccines and enzymes.

Who made the first recombinant DNA and how?

Stanley Cohen and Herbert Boyer made it in 1972. They cut an antibiotic resistance gene out of a plasmid with restriction enzymes, joined it to a native plasmid of Salmonella typhimurium with DNA ligase, and transferred it into E. coli, where it was copied by the host's DNA polymerase.

What are the steps of recombinant DNA technology in order?

The NCERT sequence is: isolation of DNA, fragmentation by restriction endonucleases, isolation of the desired fragment, ligation into a vector, transfer of the recombinant DNA into the host, large-scale culture of host cells, and extraction of the desired product. NEET often asks this order.

Why is Taq polymerase used in PCR?

Each PCR cycle begins with denaturation at a high temperature. Taq polymerase, isolated from the bacterium Thermus aquaticus, is thermostable, so it stays active through this heating and can extend the primers again in every cycle. This allows about a billion copies to be made.

Which enzymes are used to isolate DNA from different cells?

Lysozyme opens bacterial cells, cellulase plant cells and chitinase fungal cells. Ribonuclease then removes RNA and protease removes proteins such as histones. Adding chilled ethanol makes the purified DNA precipitate as fine threads, which are removed by spooling. This pure DNA can then be cut with restriction enzymes.

What is a bioreactor and why is the stirred-tank type common?

A bioreactor is a vessel in which raw materials are converted into products by cells, handling 100 to 1000 litres. The stirred-tank type is cylindrical or curved at the base, and its stirrer gives even mixing and oxygen supply, with foam, temperature and pH control and sampling ports.

What is downstream processing?

Downstream processing is the separation and purification of the product after the biosynthetic stage. The product is then formulated with suitable preservatives, tested in clinical trials as drugs are, and given strict quality control. These steps vary from product to product.

Previous year questions on Recombinant DNA Technology

42 questions from past papers, each with a step-by-step solution.

Show all 42 questions

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