Class Note

SKIE Classes Parraypora &

12th Zoology
Rajbagh
Chapter: Biotechnology Principles, Processes and Applications
Q1: What is restriction endonuclease? How does restriction endonuclease act on a DNA molecule?
Ans Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds;
exonucleases and endonucleases. Exonucleases remove nucleotides from the ends of the DNA whereas,
endonucleases make cuts at specific positions within the DNA. Each restriction endonuclease functions
by 'inspecting' the length of a DNA sequence. Once it finds its specific recognition sequence, it will bind
to the DNA and cut each of the two strands of the double helix at specific points in their sugar -phosphate
backbones. It leaves single stranded portions at the ends which forms overhanging stretches called sticky
ends on each strand. They are called sticky as they form hydrogen bonds with their complementary cut
counterparts. The stickiness of the ends facilitates the action of the enzyme DNA ligase. The steps in the
formation of recombinant DNA by action of restriction endonuclease enzyme - EcoRI is shown below

Q2: Name and explain the technique used in the separation and isolation of DNA fragments to be used
in recombinant DNA technology.
Ans: After the cutting of DNA by restriction enzyme, fragments of DNA are formed. Separation of DNA
fragments according to their size or length is done by a technique called agarose gel electrophoresis.
It is a technique of separation of molecules such as DNA, RNA or protein, under the influence of an
electric field, so that they migrate in the direction of electrode bearing the opposite charge, viz., positively
charged molecules move towards cathode (-ve electrode) and negatively charged molecules travel
towards anode (+ve electrode) through a medium/ matrix. Most commonly used matrix is agarose. DNA
Fragments separate according to their sizes through he sieving effect of agarose gel. Hence, the smaller
the: fragment size, the farther it moves. The separated DNA fragments can be seen only after staining the
DNA with a compound known as ethidium bromide (EtBr followed by exposure to UV radiation. The
fragment are visible as bright orange-coloured bands

Q3: How can DNA segments, separated by gel electrophoresis be visualised and isolated?
Ans: Ethidium bromide has the unique property of fluorescing under UV light when intercalated with DNA.
By running DNA through an ethidium bromide- treated gel (gel electrophoresis) and exposing it to UV
light, distinct bands of DNA become visible. The separated bands of DNA are cut out from the agarose

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 gel and extracted from the gel piece in a step known as elution. These fragments are then used in
constructing rDNA
Q4: Describe the characteristics a cloning vector must possess.
Ans: The following are the features that are required to facilitate cloning into a vector.
(i) Origin of replication (ori): This is a sequence from where replication starts and any piece of DNA
when linked to this sequence can be made to replicate within the host cells. This sequence is also
responsible for controlling the copy number of the linked DNA.
(ii) Selectable marker: It helps in identifying and eliminating non-transformants and selectively
permitting the growth of the transformants. Normally, the genes encoding resistance to antibiotics such
as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers
for E. coli. The normal E. coli cells do not carry resistance against any of these antibiotics.
(iii) Cloning sites: In order to link the alien DNA, the vector needs to have very few, preferably single,
recognition sites for the commonly used restriction enzymes. Presence of more than one recognition
sites within the vector will generate several fragments, which will complicate the gene cloning
Q5: Describe process of gene amplification for rDNA technology experiments.
Ans: The process of gene amplification is known as PCR. It 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 (small chemically synthesised oligonucleotides that are complementary to the regions of DNA)
and the enzyme DNA polymerase.

Each cycle of PCR involves following three steps:

(i) Denaturation: Denaturation occurs when the reaction mixture is heated to 94°C for about 15-30 minutes.
This breaks the hydrogen bonds between the two strands of DNA and converts it into a single-stranded
DNA. The single strands now act as a template for the production of new strands of DNA. The temperature
should be provided for a longer time to ensure the separation of the two strands.

(ii) Annealing: The reaction temperature is lowered to 45-60°C. Here, the primers bind to their
complementary sequences on the template DNA. Primers are single strand sequences of DNA or RNA
around 20 to 30 bases in length. These serve as the starting point for the synthesis of DNA.

(iii) Extension: At this step, the temperature is raised to 72°C. The bases are added to the 3' end of the primer
by the Taq polymerase enzyme isolated from a bacterium Thermus aquaticus. This elongates the DNA in
the 5' to 3' direction. The DNA polymerase adds about 1000bp/minute under optimum conditions. Taq
Polymerase can tolerate very high temperature. It attaches to the primer and adds DNA bases to the single
strand. As a result, a double-stranded DNA molecule is obtained

Q6: What are bioreactors? List five growth conditions that a bioreactor provides for obtaining the
desired product. Draw a well-labelled diagram of simple stirred-tank bioreactor. Also give a brief
description about it.
Ans: A bioreactor is a device in which raw materials are biologically converted into specific products by
microbes, plant/animal cells etc. These are used for food processing, fermentation, waste treatment, etc.
Growth conditions that a bioreactor provides for obtaining the desired products are:
(i) Controlled environment for optimum product yield.
(ii) Aseptic fermentation for a number of days and prevention of escape of viable cells.
(iii) Adequate mixing and aeration for optimum growth and production, without damaging the
microorganism.
(iv) Easy and dependable temperature control
(v) Facility of sampling.

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 The given figure shows simple stirred tank:

Q7: Draw a labelled diagram of sparged stirred tank bioreactor through which sterile air bubbles are
sparged duct
Ans. The labelled diagram of sparged stirred tank bioreactor is as follows:

Q8: Explain downstream processing.

Ans: 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. This process includes separation and purification
that are collectively referred to as downstream processing. The product has to be formulated with suitable
preservatives. Such formulation has to undergo thorough clinical trials as in case of drugs. Strict quality
control testing for each product is also required. The downstream processing and quality control testing
vary from product to product
Q10: (a) Name the bacterium which produces Bt toxin. What is the significance of this toxin?
(b) Name the gene which codes for Bt toxin. Why this toxin does not kill the bacterium?
(c) Name the genes that prevent infection by cotton bollworms and corn borers in pest resistant
plants.
Ans (a) Bacillus thuringiensis produces Bt toxin, an insecticidal protein. This protein kills insects such as
lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies,
mosquitoes), but it does not kill the Bacillus (bacterium) itself.
(b) Cry gene codes for this toxin. The toxin does not kill the bacterium Bacillus, as it exists in the form
of inactive protoxins. However, once an insect ingests the inactive toxin, it is converted into an active
form of toxin, due to the alkaline pH of the gut, which then creates pores in the midgut epithelial cells
and cause cell swelling, lysis and finally death of the insect. The Bt toxin protein exist as inactive
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 protoxins but once an insect ingests the inactive toxin, it is converted into an active form of toxin due
to the alkaline pH of the gut which solubilise the crystals. The activated toxin binds to the surface of
midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death
of the insect. Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into
several crop plants such as cotton. The choice of genes depends upon the crop and the targeted pest,
as most Bt toxins are insect-group specific.
(c) Proteins encoded by genes crylAc and cryIIAb control the cotton bollworms, while crylAb controls
corn borers.
Q11: Name the nematode that damages the roots of tobacco plants? How is transgenic tobacco plant
made resistant to nematode using biotechnology?
Ans: A nematode Meloidogyne incognita infects the roots of tobacco plants and causes a great reduction in
yield. A novel strategy was adopted to prevent this infestation which was based on the process of RNA
interference (RNAi). RNAi takes place in all eukaryotic organisms as a method of cellular defense. This
method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to
and prevents translation of the mRNA (silencing). The source of this complementary RNA could be
from an infection by viruses having RNA genomes or mobile genetic elements (transposons) that
replicate via an RNA intermediate. Using Agrobacterium vectors, nematode- specific genes were
introduced into the host plant. The introduction of DNA was such that it produced both sense and anti-
sense RNA in the host cells. These two RNAs being complementary to each other formed a double
stranded RNA (dsRNA) that initiated RNAi and thus, silenced the specific mRNA of the nematode. The
consequence was that the parasite could not survive in a transgenic host expressing specific interfering
RNA. The transgenic plant therefore got itself protected from the parasite.

Q12: (a) Explain the structure of human insulin with the help of a diagram.
(b) How did American company Eli Lilly produce human insulin using rDNA technique?

Ans: (a) Insulin consists of two short polypeptide chains: chain A and chain B, that are linked together by
disulphide bridges. In mammals, including humans, insulin is synthesised as a prohormone which
contains an extra stretch called the C peptide. This C peptide is not present in the mature insulin and is
removed during maturation into insulin. The given diagram explains the maturation of proinsulin into
insulin
(b) In 1983, Eli Lilly, an American company first prepared two DNA sequences corresponding to A and
B chains of human insulin and introduced them in plasmids of Escherichia coli to produce insulin chains.
Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form
human insulin (Humulin). It is recombinant DNA technological process

Q13: (a) What is gene therapy? Illustrate using the example of adenosine deaminase (ADA) deficiency:
(b) Name any other possible treatments for ADA deficiency:

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 Ans: (a) Gene therapy is the technique of genetic engineering to replace a faulty gene by a normal healthy
functional gene. The first clinical gene therapy was given in 1990 to a 4 years old girl with adenosine
deaminase deficiency (ADA deficiency). This enzyme is very important for the immune system to
function. Severe combined immunodeficiency (SCID) is caused due to defect in the gene for the enzyme
adenosine deaminase. SCID patient lacks functional T-lymphocytes and therefore, fails to fight the
infecting pathogens. Lymphocytes are extracted from the patient's bone marrow and a normal functional
copy of human gene coding for ADA is introduced into these lymphocytes with the help of retroviral
vector. The cells so treated are reintroduced into the patient's bone marrow. The lymphocytes produced
by these cells contain functional ADA gene which reactivate the victim's immune system. Though these
cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes.
However, if the gene isolated from marrow cells producing ADA is introduced into cells at early
embryonic stages, it could be a permanent cure. Steps of gene therapy can be summarised in the given
diagram:
(b) Other possible treatments that can be given to a patient exhibiting adenosine deaminase (ADA)
deficiency is bone marrow transplantation and enzyme replacement therapy:

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