Chapter–11

Biotechnology:
Principles and
Processes

1. Biotechnology
QQ Biotechnology deals with microorganisms, plant or animal cells or their enzymes to produce products
and processes useful to humans.
QQ The term “Biotechnology” was given by Karl Ereky (1919).
QQ According to European Federation of Biotechnology (EFB), biotechnology is the integration of natural
science and organisms, cells, parts thereof, and molecular analogues for products and services.

2. Principles of Biotechnology
QQ The two core techniques that developed modern biotechnology are:
(i) Genetic engineering which is modification of chemical nature of DNA/RNA and their
introduction into another host organism, to change the phenotypic characters of the host.
(ii) Sterilisation methods to maintain growth and manipulation of only the desired microbes
or cells in large quantities, for the manufacture of biotechnological products like antibiotics,
vaccines, enzymes, etc.
QQ The basic steps in genetic engineering include:
(i) identification of DNA with desirable genes.
(ii) introduction of the DNA into host to form recombinant DNA (rDNA).
(iii) maintenance of DNA in host and gene cloning.
(iv) gene transfer.
QQ In 1972, Stanley Cohen and Herbert Boyer constructed the first recombinant DNA.
QQ Herbert Boyer worked on restriction enzymes of E. coli which cut DNA in particular fashion and
produce sticky ends on both the strands. These restricted ends were ligated with desired pieces of
DNA.
QQ Stanley Cohen studied plasmid DNA floating freely in cytoplasm of bacterial cells.. He also developed
a method of removing plasmids from the cell and reinserting them in other cells.
QQ They isolated antibiotic resistant gene from plasmid of bacteria and then linked the gene with plasmid
and incorporated into E coli, where it could replicate using the new host’s DNA polymerase enzyme
and make multiple copies.
QQ Steps carried out in constructing first recombinant DNA:
(i) A gene encoding antibiotic resistance in the native plasmid of Salmonella typhimurium V. was
identified. Plasmid is an autonomously replicating circular extra-chromosomal DNA.

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 (ii) The desired DNA was cut at specific locations by restriction enzymes.
(iii) The cut DNA was linked to plasmid DNA and transferred to E. coli for gene multiplication.

3. Tools of Recombinant DNA Technology

QQ The key tools required for the recombinant DNA technology are:
(i) Restriction enzymes (ii) Polymerase enzymes
  (iii) Ligases (iv) Vectors
(v) Host organism/cell.

4. Restriction Enzymes
QQ The restriction enzymes are called “molecular scissors” and are responsible for cutting DNA.
QQ They are present in bacteria to provide a type of defence mechanism called the “restriction–
modification system”.
QQ The first restriction endonuclease, HindII, was isolated by Smith, Wilcox and Kelley (1968) from
Haemophilus influenzae bacterium. It was used to cut DNA molecules at a particular point by
recognising a specific sequence of six base pairs, known as the recognition sequence.

Naming of Restriction Enzymes

QQ The first letter is derived from the genus name and the next two letters from the species name of the
prokaryotic cell from where the enzymes are extracted. The roman numbers, following the name,
show the order in which the enzymes were isolated from the bacterial strain.
QQ For example, EcoRI is derived from Escherichia coli RY13, HindII from Haemophilus influenzae Rd,
BamHI from Bacillus amyloliquefaciens H, EcoRII from E. coli R245, etc.
QQ Restriction enzymes belong to a class of enzymes called nucleases and are of two types:
(i) Exonucleases—cut DNA at the ends.
(ii) Endonucleases—cut at specific positions within the DNA.
QQ The recognition sequences of endonucleases are palindromic, i.e., the sequence of base pairs read the
same on both the DNA strands, when orientation of reading is kept same, e.g.,
5′ GAATTC 3′
3′ CTTAAG 5′

Mechanism of Action of Endonucleases

QQ Every endonuclease inspects the entire DNA sequence for the palindromic recognition sequence.
QQ On finding the palindrome, the endonuclease binds to the DNA.
QQ It cuts the opposite strands of DNA in the sugar–phosphate backbone; a little away from the centre of
the palindrome sites but between the same bases on both strands.
QQ This results in the formation of single stranded overhanging stretches at the end of each strand called
sticky ends.
QQ The sticky ends facilitate the action of the enzyme DNA ligase by readily forming hydrogen bonds
with complementary strands.
QQ In genetic engineering, DNA from different sources are cut with the same restriction enzymes so that
both DNA fragments have same kind of sticky ends.
QQ These sticky ends are complementary to each other and thus can be joined by DNA ligase (end-to-
end).

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 Fig. 11.1 Steps in formation of recombinant DNA by action of restriction
endonuclease enzyme EcoRI

5. Separation and Isolation of DNA Fragments (Gel Electrophoresis)

QQ Gel electrophoresis is a technique for separating DNA fragments based on their size.
QQ Firstly, the sample DNA is cut into fragments by restriction endonucleases.

Cathode (–ve)
Anode (+ve)

Fig. 11.2 A typical agarose gel electrophoresis showing migration of undigested (lane 1) and
digested set of DNA fragments (lane 2 to 4)

QQ The DNA fragments being negatively charged can be separated by forcing them to move towards the
anode under an electric field through a medium/matrix.
QQ Commonly used matrix is agarose, which is a natural linear polymer of D-galactose and 3, 6-anhydro-
L-galactose which is extracted from sea weeds.
QQ The DNA fragments separate-out (resolve) according to their size because of the sieving property of
agarose gel. Hence, smaller the fragment size, the farther it will move.
QQ The separated DNA fragments are visualised after staining the DNA with ethidium bromide
followed by exposure to UV radiation.
QQ The DNA fragments are seen as orange coloured bands.
QQ The separated bands of DNA are cut out and extracted from the gel piece. This step is called elution.
QQ The purified DNA fragments are used to form recombinant DNA which can be joined with cloning
vectors.

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6. Cloning Vectors
QQ The vectors are the DNA molecules that can carry a foreign DNA segment into the host cell.
QQ Vectors may be:
(a) Plasmids: These are autonomously replicating circular extra-chromosomal DNA.
(b) Bacteriophages: These are viruses that infect bacteria. Bacteriophages because of their high
number per cell have very high copy number within bacterial cells.
QQ Copy number: It is defined as the number of copies of vectors present in a cell. It varies from
1–100 copies per cell.
QQ The best known vector is the plasmid vector.
QQ pBR322 is the first artificial cloning vector developed in 1977 by Boliver and Rodriguez from
E. coli plasmid.
QQ The following features are required to facilitate cloning into a vector:

(i) Origin of replication (ori)

OO This is a DNA sequence that is responsible for
initiating replication. Any piece of DNA when linked
to this sequence can replicate within the host cells.
OO ori also controls the copy numbers of the linked
DNA. For many copies of target DNA, it should be
cloned in a vector whose origin supports high copy
number.

(ii) Selectable marker rop

OO It helps to select the host cells which contain the
vector (transformants) and eliminate the non-
transformants.
OO Transformation is defined as the procedure by which Fig. 11.3. E. coli cloning vector pBR322
showing restriction sites (HindIII, EcoRI,
a piece of DNA is introduced into a bacterial cell. BamHI, SalI, PvuII, PstI, ClaI), ori and
OO Genes encoding resistance to antibiotics like antibiotic resistance genes (ampR and tetR).
ampicillin, chloramphenicol, tetracycline or rop codes for the proteins involved in the
kanamycin, are useful selectable markers for E. coli. replication of the plasmid.
OO The normal E. coli cells do not carry resistance against any of these antibiotics.

(iii) Cloning sites

OO To link the alien DNA, the vectors require very few (mostly single) recognition sites for the
restriction enzymes.
OO More than one recognition sites within the vector, can complicate the gene cloning as it will
generate several fragments.
OO Ligation of alien DNA can be carried out at a restriction site present in one of the two antibiotic
resistance genes.

(iv) Vectors for cloning genes in plants and animals

OO There are several vectors which are used for cloning genes in plants and animals.
OO In plants, the tumour inducing plasmid (Ti) of Agrobacterium tumefaciens is used as a cloning vector.
OO A. tumefaciens is a pathogen of several dicot plants.
OO It delivers a piece of DNA known as ‘T-DNA’ in the Ti plasmid which transforms normal plant
cells into tumor cells to produce chemicals against pathogens.

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 OO Retrovirus, adenovirus, papillomavirus are also now used as cloning vectors in animals because
of their ability to transform normal cells into cancerous cells. They are first disarmed and then
used to transfer desirable genes to animal cells.
QQ Selection of recombinants formed can be done by one of the following methods:
(a) Inactivation of antibiotics
OO If a foreign DNA ligates at the BamHI site of tetracycline resistance gene in the vector pBR322, the
recombinant plasmid loses the tetracycline resistance due to insertion of foreign DNA.
OO It can still be selected out from non-recombinant ones by plating the transformants on ampicillin
containing medium.
OO The transformants growing on ampicillin containing medium are then transferred on to a medium
containing tetracycline.
OO The recombinants can grow in ampicillin containing medium but not on that containing
tetracycline whereas non-recombinants can grow on the medium containing both the antibiotics
and thus recombinants are selected.
(b) Alternative Selectable Marker (Insertional inactivation)
OO On the basis of colour production in the presence of chromogenic substrate, the recombinants
and non-recombinants can also be differentiated.
OO Here, a recombinant DNA is inserted within the coding sequence of an enzyme β-galactosidase,
which results into inactivation of the enzyme and hence there is no conversion of substrate to
products.
OO Hence, the bacterial colonies having transformed plasmid, shows no colouration while those
without inserted plasmid form blue colour colonies.

7. Competent Host (For Transformation with Recombinant DNA)

QQ DNA being a hydrophilic molecule, cannot pass through cell membranes.
QQ Therefore, the bacteria should be made competent to accept the DNA molecules.
QQ Competency is the ability of a cell to take up foreign DNA.
QQ The cell is made competent by the following methods:
(i) Chemical method (ii) Physical method

(i) Chemical method

OO The cell is treated with specific concentration of a divalent cation such as calcium to increase pore
size in cell wall.
OO The cells are incubated with recombinant DNA on ice, followed by placing them briefly at 42°C
and then putting it back on ice. This is called heat shock treatment.
OO The bacteria now take up the recombinant DNA.

(ii) Physical methods

The physical methods include
OO Micro-injection method: Recombinant DNA is directly injected into the nucleus of an animal cell
of micro-pipettes.
OO Biolistic or gene gun method: Cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA in plants.
QQ Disarmed pathogen vectors are also used to transfer rDNA like Agrobacterium tumefaciens.

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8. Process of Recombinant DNA Technology
QQ Recombinant DNA technology involves the following steps:
(i) Isolation of DNA
(ii) Fragmentation of DNA by restriction endonucleases
(iii) Isolation of a desired DNA fragment
(iv) Amplification of the gene of interest
(v) Ligation of the DNA fragment into a vector
(vi) Insertio­n of recombinant DNA into the host
(vii) Culturing the host cells on a suitable medium at a large scale
(viii) Extraction of the desired gene product
(ix) Downstream processing of the products as finished product, ready for marketing

Foreign DNA Vector

DNA
Same restriction enzyme cutting both foreign (Plasmid)
DNA and vector DNA at specific point

Ligases join foreign

DNA to plasmid

Transformation

E. coli
Cells divide

Fig. 11.4 Diagrammatic representation of recombinant DNA technology

(i) Isolation of the genetic material (DNA)

OO To cut DNA with restriction enzymes DNA should be purified, free from other macromolecules.
OO The bacterial/plant/animal cell is broken down by enzymes to release DNA, along with RNA,
proteins, polysaccharides and lipids.
OO Bacterial cell is treated with enzyme lysozyme.
OO Plant cell is treated with enzyme cellulase.
OO Fungal cell is treated with chitinase.

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 OO RNA is removed by treatment with ribonuclease and proteins are removed by treatment with
protease.
OO After several treatments, the purified DNA is precipitated by adding chilled ethanol.

(ii) Cutting of DNA at specific locations

OO The DNA is cut using restriction enzymes.
OO The purified DNA is incubated, with the specific restriction enzyme at conditions optimum for
the enzyme to act.
OO Process is repeated with vector DNA also.

(iii) Isolation of desired DNA fragment

OO Using agarose gel electrophoresis, the activity of the restriction enzymes can be checked.
OO Since the DNA is negatively charged, it moves towards the positive electrode or anode and in the
process, DNA fragments separate out based on their sizes.
OO The desired DNA fragment is eluted out.

(iv) Amplification of gene of interest using PCR

OO The Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific DNA
sequences is carried out in vitro.
OO This technique was developed by Kary Mullis in 1985, and for this, he received Nobel Prize for
Chemistry in 1993.

Fig. 11.5. Polymerase chain reaction (PCR)

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 OO Requirements for PCR:

(a) DNA template: The double-stranded DNA that needs to be amplified.

(b) Primers: Small chemically synthesised oligonucleotides of about 10–18 nucleotides that are
complementary to a region of template DNA.
(c) Enzyme: Two commonly used enzymes are Taq polymerase (isolated from thermophilic
bacterium, Thermus aquaticus) and Vent polymerase (isolated from Thermococcus litoralis).
OO PCR is carried out in the following three steps:

(a) Denaturation
— The double-stranded DNA is denatured by subjecting it to high temperature of 94°C for 15
seconds. Each separated single stranded strand now acts as template for DNA synthesis.
(b) Annealing
— Two sets of primers are added which anneal to the 3′ end of each separated strand.
— Primers act as initiators of replication.
(c) Extension
— DNA polymerase extends the primers by adding nucleotides complementary to the template
provided in the reaction.
— A thermostable DNA polymerase (Taq polymerase) is used in the reaction which can tolerate
the high temperature of the reaction.
— All these steps are repeated many times to obtain several copies of desired DNA.

(v) Ligation of DNA fragment into a vector

OO The vector DNA and source DNA are cut with the same endonuclease to obtain sticky ends.
OO These are then ligated by mixing vector DNA, gene of interest and enzyme DNA ligase to form
a recombinant DNA.

(vi) Insertion of recombinant DNA into the host cell/organism

OO Introduction of ligated DNA into recipient cells occurs by several methods, before which the
recipient cells are made competent to receive the DNA.
OO If recombinant DNA carrying antibiotic resistance (e.g., ampicillin) is transferred into E. coli cells,
the host cell is transformed into ampicillin-resistant cells.
OO The ampicillin resistant gene in this case is called a selectable marker.
OO On growing transformed cells on agar plates containing ampicillin, only transformants will grow
and others will die.

(vii) Culturing the host cells

OO The transformed host cells are grown in appropriate nutrient medium at optimal conditions.
OO The DNA gets multiplied and expresses itself to form desired product.

(viii) Extraction of desired gene product

OO When a protein encoding gene is expressed in a heterologous host, it is called a recombinant
protein.
OO The cells having genes of interest can be grown on a small scale or on a large scale.
OO On small scale, the cells are grown on cultures in laboratory and then the expressed protein is
extracted and purified by different separation techniques.
OO On large scale, the cells are grown in a continuous culture system in which fresh medium is
added from one side, to maintain cells in exponential growth phase and the desired protein is
collected from the other side.
OO In large scale method, larger biomass is produced which leads to high yield.

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 (ix) Downstream processing
OO All the processes to which a product is subjected to before being marketed as a finished product
are called downstream processing.
OO It includes:
(a) Separation of the product from the reactor.
(b) Purification of the product.
(c) Formulation of the product with suitable preservatives.
(d) Quality control testing and clinical trials in case of drugs.

9. Bioreactors
QQ Bioreactors are vessels of large volumes (100–1000 litres) in which raw materials are biologically
converted into specific products.
QQ It provides all the optimal conditions for achieving the desired product by providing optimal growth
conditions like temperature, pH, substrates, salts, vitamins and oxygen.
QQ Stirred-tank bioreactors are commonly used bioreactors.

air
(a) (b)

Fig. 11.6. (a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through which sterile air
      bubbles are sparged.
QQ These are cylindrical with curved base to facilitate (i) proper mixing of the contents, (ii) maintain
oxygen availability throughout the bioreactor.
QQ Stirred tank reactor has (i) better temperature and pH control, (ii) foam control system to prevent
foam and shearing damage to cells due to agitation, (iii) system sterilisation, and (iv) provision to
withdraw small volumes of cultures periodically.
QQ A bioreactor has the following components:
(i) An agitator system
(ii) An oxygen delivery system
(iii) Foam control system
(iv) Temperature control system
(v) pH control system
(vi) Sampling ports to withdraw cultures periodically
QQ The stirrer mixes the contents and makes oxygen available throughout the bioreactor.
QQ Sparged stirred-tank reactor is a stirred type reactor in which air is bubbled.

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NCERT Textbook Questions
Q. 1. Can you list 10 recombinant proteins which are used in medical practice? Find out where they
are used as therapeutics (use the internet).
Ans.
S. No. Recombinant proteins Therapeutic uses
(i) Human Insulin (Humulin) Treatment of diabetes type 1.
(ii) Tissue Plasminogen Activator Treatment for acute myocardial infarction; dissolves
blood clot after heart attack and stroke.
(iii) DNase Treatment of cystic fibrosis.
(iv) Platelet Growth Factor Stimulation of wound healing.
(v) Calcitonin Treatment of rickets.
(vi) Reo Pro Prevention of blood clots.
(vii) Hirudin Used as an anticoagulant.
(viii) Interferon (α, β and γ) Treatment of viral infection and cancer.
(ix) Chorionic Gonadotropin Treatment of infertility.
(x) Interleukins Enhancing activity of immune system.

Q. 2. Make a chart (with diagrammatic representation) showing a restriction enzyme, the substrate
DNA on which it acts, the site at which it cuts DNA and the product it produces.
Ans. Refer to Fig. 11.1.
Q. 3. From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in
molecular size? How did you come to know? [HOTS]
Ans. DNA is bigger in molecular size. DNA is made up of sugar, phosphate and nitrogenous bases. An
enzyme is made up of only protein (one or few polypeptides), so there is no complexity of molecules.
Q. 4. What would be the molar concentration of human DNA in a human cell? Consult your teacher.
[HOTS]
Ans. The average molecular weight of a nucleotide pair is 330 dalton. Genome size of a diploid human
cell is around 6 × 109 bp. The concentration of DNA will be 330 × 6 × 109g/mol.
The molarity can then be calculated as:
Sample DNA concentration (in g)

330 × 6 × 10 9 g/mol
Q. 5. Do eukaryotic cells have restriction endonucleases? Justify your answer. [HOTS]
Ans. Eukaryotic cells have no restriction enzymes as the DNA molecules of eukaryotes are heavily
methylated. It is present in prokaryotic cell (like bacteria) where these act as defence mechanism
to restrict the growth of bacteriophages.
Q. 6. Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors
have over shake flasks?
Ans. Other advantages of stirred tank bioreactors over shake flasks are that these facilitate temperature
control system, pH control system, foam control system and sampling ports from where small
volume of the cultures can be obtained and tested time to time.
Q. 7. Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to
create a palindromic sequence by following base-pair rules.
Ans. (i) 5′ G A A T T C 3′ (ii) 5′ G G A T C C 3′
3′ C T T A A G 5′ 3′ C C T A G G 5′
(iii) 5′ A C T A G T 3′ (iv) 5′ A A G C T T 3′
3′ T G A T C A 5′ 3′ T T C G A A 5′
(v) 5′ A G G C C T 3′
3′ T C C G G A 5′

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 Q. 8. Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Ans. A recombinant DNA is made during pachytene stage of meiosis-I by crossing over.
Q. 9. Can you think and answer how a reporter enzyme can be used to monitor transformation of
host cells by foreign DNA in addition to a selectable marker? [HOTS]
Ans. A reporter gene encodes an enzyme, with an easily transcriptional activity of a gene of interest.
A reporter gene is the one whose phenotypic expression can be monitored and thus it reports
about activity or change in advance of the effect of modification, in addition to eliminating
non-transformed cells by selectable markers.
Q. 10. Describe briefly the following:
(a) Origin of replication (b) Bioreactors (c) Downstream processing
Ans. (a) Origin of replication is a DNA sequence that initiates any piece of linked DNA to replicate
and is also called ori site. It controls the copy numbers of the linked DNA.
(b) Refer to Basic Concepts Point 9.
(c) Refer to Basic Concepts Point 8 (ix).
Q. 11. Explain briefly
(a) PCR (b) Restriction enzymes and DNA  (c) Chitinase
Ans. (a) PCR stands for Polymerase Chain Reaction, which is a method for amplification of small
segments of DNA.
(b) Restriction enzymes are also called ‘molecular scissors’ because they cut the helix of DNA at
a specific site. DNA is the genetic material, which carries and pass the genetic characters or
information from one generation to other.
(c) Chitinase is an enzyme which is used to cut or break the cell wall of fungi to release its
cellular components.
Q. 12. Discuss with your teacher and find out how to distinguish between
(a) Plasmid DNA and chromosomal DNA (b) RNA and DNA
(c) Exonuclease and endonuclease
Ans. (a) Table 11.1: Differences between Plasmid DNA and Chromosomal DNA
S. No. Plasmid DNA Chromosomal DNA
(i) This is present in prokaryotic cells (bacteria). This is present in both prokaryotic and
eukaryotic cells.
(ii) This is the circular extra-chromosomal DNA It is linear and associated with histones
not associated with histone proteins. proteins in eukaryotes but is double
stranded and circular in prokaryotes.
(iii) It gives the cell extra characters like antibiotic It contains genes for characters essential for
resistance. life of organism.

(b) Table 11.2: Differences between DNA and RNA

S. No. DNA RNA
(i) It has deoxyribose sugar. It has ribose sugar.
(ii) It is the genetic material in almost all It is the genetic material in only some viruses.
organisms.
(iii) It is double stranded. It is single stranded.
(iv) It has A, G, C, T bases. It has A, G, C, U bases.

(c) Table 11.3: Differences between Exonuclease and Endonuclease

S. No. Exonuclease Endonuclease
(i) These cut the end regions of the DNA. These cut at specific regions within the DNA.
(ii) These act on single strand of DNA. These act on both strands as well as on DNA
strand.

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Multiple Choice Questions [1mark]
Choose and write the correct option in the following questions.
1. Restriction endonuclease
(a) synthesizes DNA
(b) cuts the DNA molecues randomly
(c) cuts the DNA molecule at specific sites
(d) restricts the synethesis of DNA inside the nuclease
2. The linking of antibiotic resistance gene with the plasmid vector became possible with
(a) DNA polymerase (b) exonucleases
(c) DNA ligase (d) endonucleases
3. Stirred-tank bioreactors have been designed for
(a) addition of preservatives to the product
(b) purification of the product
(c) ensuring anaerobic conditions in the culture vessel
(d) availability of oxygen throughout the process
4. Given below is a sample of a portion of DNA strand giving the base sequence on the opposite
strands. What is so special shown in it?
5′_____GAATTC_____3′
3′_____CTTAAG_____5′
(a) Replication completed (b) Deletion mutation
(c) Start codon at the 5′ level (d) Palindromic sequence of base pairs
5. There is a restriction endonuclease called Eco RI. What does “co” part in it stand for?
(a) Colon (b) Coelom
(c) Coenzyme (d) Coli
6. Agarose extracted from sea weeds is used in
(a) spectrophotometry (b) tissue culture
(c) PCR (d) gel electrophoresis
7. An enzyme catalysing the removal of nucleotides from the ends of DNA is [NCERT Exemplar]
(a) endonuclease (b) exonuclease
(c) DNA ligase (d) Hind-II
8. The transfer of genetic material from one bacterium to another through the mediation of a
viral vector is termed as: [NCERT Exemplar]
(a) transduction (b) conjugation
(c) transformation (d) translation
9. Which of the given statements is correct in the context of observing DNA separated by agarose
gel electrophoresis? [NCERT Exemplar]
(a) DNA can be seen in visible light
(b) DNA can be seen without staining in visible light
(c) Ethidium bromide stained DNA can be seen in visible light
(d) Ethidium bromide stained DNA can be seen under exposure to UV light
10. Which of the following is not a characterstic of the plasmids ?
(a) Extranuclear (b) Single-stranded
(c) Independent replication (d) Circular DNA

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 11. DNA fragments generated by the restriction endonuclease in a chemical reaction can be
separated by
(a) Gel electrophoresis (b) Restriction mapping
(c) Centrifugation (d) PCR
12. Which of the following is not required in the preparation of a recombinant DNA molecule?
[NCERT Exemplar]
(a) Restriction endonuclease (b) DNA ligase
(c) DNA fragments (d) E.coli
13. In agarose gel electrophoresis, DNA molecules are separated on the basis of their
[NCERT Exemplar]
(a) charge only (b) size only
(c) charge to size ratio (d) All of the above
14. The most important feature in a plasmid to be used as a vector is [NCERT Exemplar]
(a) origin of replication (ori) (b) presence of a selectable marker
(c) presence of sites for restriction endonuclease(d) its size
15. While isolating DNA from bacteria, which of the following enzymes is not required?
[NCERT Exemplar]
(a) Lysozyme (b) Ribonuclease
(c) Deoxyribonuclease (d) Protease
16. Which of the following has popularised the PCR (polymerase chain reactions)?
[NCERT Exemplar]
(a) Easy availability of DNA template
(b) Availability of synthetic primers
(c) Availability of cheap deoxyribonucleotides
(d) Availability of ‘Thermostable’ DNA polymerase
17. Select the correct sequence of processing of PCR.
(a) Extension, primer annealing, denaturation
(b) Denaturation, primer annealing, extension
(c) Denaturation, extension, primer annealing
(d) Primer annealing, denatturatiotn, extension
18. Which of the following is/are used in recombinant DNA technology ?
1. Agarose gel 2. Restriction endonuclease
3. Plasmid vector 4. Ethidium bromide
(a) 1 and 2 (b) 2 and 3
(c) 3 and 4 (d) All of these
19. An antibiotic resistance gene in a vector usually helps in the selection of [NCERT Exemplar]
(a) competent cells (b) transformed cells
(c) recombinant cells (d) none of the above
20. Significance of ‘heat shock’ method in bacterial transformation is to facilitate
[NCERT Exemplar]
(a) binding of DNA to the cell wall
(b) uptake of DNA through membrane transport proteins
(c) uptake of DNA through transient pores in the bacterial cell wall
(d) expression of antibiotic resistance gene

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 21. The role of DNA ligase in the construction of a recombinant DNA molecule is [NCERT Exemplar]
(a) formation of phosphodiester bond between two DNA fragments
(b) formation of hydrogen bonds between sticky ends of DNA fragments
(c) ligation of all purine and pyrimidine bases
(d) None of the above
22. Which of the following steps are catalysed by Taq DNA polymerase in a PCR reaction?
[NCERT Exemplar]
(a) Denaturation of template DNA
(b) Annealing of primers to template DNA
(c) Extension of primer end on the template DNA
(d) All of the above
23. A bacterial cell was transformed with a recombinant DNA molecule that was generated using
a human gene. However, the transformed cells did not produce the desired protein. Reasons
could be: [NCERT Exemplar]
(a) Human gene may have intron which bacteria cannot process
(b) Amino acid codons for humans and bacteria are different
(c) Human protein is formed but degraded by bacteria
(d) All of the above
24. In genetic engineering, the antibiotics are used
(a) as selectable markers (b) to keep the cultures free of inection
(c) to select healthy vectors (d) as sequence from where replication starts
25. Which of these is not correctly matched?
(a) Gene gun—biolistic gun (b) Plasmids—extrachromosomal DNA
(c) DNA ligase—biological scissors (d) Bacteriophages—viruses
26. Which of the following statements does not hold true for restriction enzyme? [NCERT Exemplar]
(a) It recognises a palindromic nucleotide sequence
(b) It is an endonuclease
(c) It is isolated from viruses
(d) It can produce the same kind of sticky ends in different DNA molecules

Answers
1. (c) 2. (c) 3. (d) 4. (d) 5. (d) 6. (d) 7. (b) 8. (a) 9. (d) 10. (b)
11. (a) 12. (d) 13. (d) 14. (a) 15. (c) 16. (d) 17. (b) 18. (d) 19. (b) 20. (c)
21. (a) 22. (c) 23. (a) 24. (a) 25. (c) 26. (c)

Assertion-Reason Questions
In the following questions a statement of assertion followed by a statement of reason is given. Choose
the correct answer out of the following choices.
(a) Assertion and reason both are correct statements and reason is correct explanation for assertion.
(b) Assertion and reason both are correct statements but reason is not correct explanation for
assertion.
(c) Assertion is correct statement but reason is wrong statement.
(d) Assertion is wrong statement but reason is correct statement.
1. Assertion : Plasmids are single stranded extrachromosomal DNA.
Reason : Plasmids are found in eukaryotic cells.
2. Assertion : Plasmids are extrachromosomal DNA.
Reason : Plasmids are found in bacteria and are useful in genetic engineering.

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 3. Assertion : In recombinant DNA technology, human genes are often transferred into bacteria
(prokaryotes) or yeast (eukaryote).
Reason : Both bacteria and yeast multiply very fast to form huge population which express
the desired gene.
4. Assertion : Insertion of recombinant DNA within the coding sequence of beta-galactosidase
results in colourless colonies.
Reason : Presence of insert results in inactivation of enzyme beta-galactosidase known as
insertional inactivation.
5. Assertion : In recombinant DNA technology both ligase and nuclease play an important role.
Reason : Ligase cuts the DNA at specific sites and nuclease joins the DNA fragments.
6. Assertion : E.coli having pBR322 with DNA insert at BamHI site cannot grow in medium
containing tetracycline.
Reason : Recognition site for BamHI is present in tetr region of pBR322.
7. Assertion : Downstream processing include separation and purification of product.
Reason : Before release of the product, it needs to be tested for quality control.
8. Assertion : PCR primers do not have self complementary regions.
Reason : PCR involves use of Taq polymerase as it can withstand the high temperature of
the process.
9. Assertion : Restriction enzymes recognise palindromic sequences.
Reason : Palindromic sequences read the same in both directions of the strands.
10. Assertion : For isolating DNA from yeast cell, chitinase enzyme is necessary.
Reason : Cell wall of fungi are made up of chitin.

Answers
1. (c) 2. (a) 3. (a) 4. (a) 5. (c) 6. (a) 7. (b) 8. (d) 9. (b) 10. (a)

Case-based/Source-based Question
1.
(i) Name the organism in which the vector shown is inserted to get the copies of the desired
gene.
(ii) Mention the area labelled in the vector responsible for controlling the copy number of the
inserted gene.
(iii) Name and explain the role of a selectable marker in the vector shown.

rop

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 Ans. (i) Escherichia coli.
(ii) Origin of replication or ‘ori’ controls copy number of inserted gene.
(iii) The selectable markers are ampR and tetR (resistance to ampicillin, tetracycline). Selectable
markers help to select the host cells which contain the vector (transformants) and eliminate
non-transformants. If a foreign DNA ligates at the BamHI site of tetracycline resistance
gene in the vector pBR322, the recombinant plasmid loses the tetracycline resistance due to
insertion of DNA. It can still be selected out from non-recombinant.
2. Rajesh was doing gel electrophoresis to purify DNA fragments. Given below is the sketch of
the observations of the experiment performed by him.

A
B

(i) At which end he would have loaded the samples and where?
(ii) Analyse the reason for different positions taken up by the DNA bands.
(iii) Elaborate the step he would have followed to visualise DNA bands.
Ans. (i) He would have loaded the samples near end A; in the wells.
(ii) The DNA fragments separate (resolve) according to their size through sieving effect provided
by the agarose gel. Hence, the smaller the fragment size, the farther it moves.
(iii) After staining the DNA with ethidium bromide followed by exposure to UV radiations the
DNA bands appear coloured.
3. Study the diagram given below and answer the questions that follow:

(i) What is EcoRI?

(ii) How is the action of exonuclease different from that of endonuclease?
(iii) How are ‘sticky ends’ formed on a DNA strand? Why are they so called?

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 Ans. (i) EcoRI is a restriction endonuclease enzyme.
(ii) Exonucleases cleave the DNA molecules at their ends whereas endonucleases cleave DNA
molecules internally.
(iii) Restriction enzymes cut the strands of the DNA, a little away from the centre of the palindromic
sites, but between the same two bases on opposite strands. This leaves sticky single stranded
position at the ends. These overhanging stretches are aids. These are named so because they
form hydrogen bonds with their complementary cut counterparts very easily.
4. Observe the diagram of the first artificial plasmid vector pBR322.
Cla c
b

Pvu
Bam
Pst
a d

Sal

rop

Pvu
(i) Identify the selectable markers in the diagram of E. coli vector shown above.
(ii) How is the coding sequence of β-galactosidase considered a better marker than the ones
identified by you in the diagram? Explain.
(iii) Why is it essential to have a ‘selectable marker’ in a cloning vector? [CBSE (AI) 2011]
Ans. (i) a—gene for ampicillin resistance
d—gene for tetracycline resistance.
(ii) The insertion of rDNA into the coding sequence of an enzyme β-galactosidase leads to the
inactivation of the enzyme. This is called insertional inactivation. The recombinants do not
produce blue-coloured colonies in the presence of chromogenic substrate while the non-
recombinants produce a blue colour. Thus, coding sequence of β-galactosidase is a better
marker.
(iii) Selectable markers are essential to identify and eliminate non-transformants, by selectively
permitting the growth of the transformant.
5. Bioreactors are vessels for production of large-scale gene products.

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 Answer the following questions based on the above information.
(i) How has the development of bioreactors helped in biotechnology?
(ii) What are recombinant proteins?
(iii) How do bioreactors help in their production?
Ans. (i) In bioreactors large volume of culture can be processed which results in higher yields of
the desired specific products (protein/enzyme). The entire process takes place under the
controlled conditions of temperature, pH and raw materials.
(ii) The protein produced by genetically altered gene in a host is called recombinant protein.
Bioreactors are vessels in which raw materials are biologically converted into specific
products by microbes.
(iii) It provides optimum growth conditions such as temperature, pH, substrate, vitamins, oxygen
and salts.

Very Short Answer Questions [1 mark]

Q. 1. Write the two components of the first artificial recombinant DNA molecule constructed by
Cohen and Boyer. [CBSE (F) 2014, CBSE Sample Paper 2018]
Ans. The two components were—antibiotic resistance gene and plasmid vector of Salmonella
typhimurium.
Q. 2. What is the host called that produces a foreign gene product? What is this product called?
[CBSE (F) 2010]
Ans. The host that produces a foreign gene product is called competent host. The product is called
recombinant protein.
Q. 3. Give any two microbes that are useful in biotechnology. [NCERT Exemplar]
Ans. E. coli and Saccharomyces cerevisiae.
Q. 4. Mention the uses of cloning vector in biotechnology. [CBSE Delhi 2011]
Ans. Cloning vectors are used for transferring fragments of foreign DNA into a suitable host. They are
also used to select recombinants from non-recombinants.
Q. 5. What is the function of restriction enzyme?
Ans. To cut DNA at specific site.
Q. 6. Name the first plasmid used as vector.
Ans. pBR322.
Q. 7. What are palindromes?
Ans. Palindromes are group of letters (sequences) that read same both in forward and backward
direction.
Q. 8. What is recombinant DNA?
Ans. Recombinant DNA is the DNA formed by combining DNAs from two different organisms.
Q. 9. Biotechnologists refer to Agrobacterium tumifaciens as a natural genetic engineer of plants.
Give reasons to support the statement. [CBSE (AI) 2011] [HOTS]
Ans. This is because A. tumifaciens can transfer genes naturally by delivering a piece of T-DNA to plant
cells. It has a tumour inducing plasmid.
Q. 10. Why is the enzyme cellulase needed for isolating genetic material from plant cells and not
from the animal cells? [CBSE Delhi 2010, 2013] [HOTS]
Ans. The enzyme cellulase breaks down cellulose which is present in cell walls of plants but absent in
animal cells.
Q. 11. Name the compound used for staining the isolated DNA in the gel electrophoresis.
Ans. Ethidium bromide.

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Q. 12. Why does DNA move towards the anode in gel electrophoresis? [HOTS]
Ans. The DNA fragments are negatively charged so they move towards the positively charged anode.
Q. 13. Suggest a technique to a researcher who needs to separate fragments of DNA. [CBSE Delhi 2016]
Ans. Gel electrophoresis is used to separate DNA fragments.
Q. 14. What is gene gun?
Ans. The instrument for bombarding micro-projectile particles (gold/tungsten particles) coated with
foreign DNA, with great velocity, into a target cell is called gene gun.
Q. 15. How does an alien DNA gain entry into a plant cell by ‘biolistics’ method? [CBSE (F) 2013]
Ans. In biolistics method, cells are bombarded with high velocity micro-particles of gold or tungsten
coated with DNA.
Q. 16. Why EtBr is used in gel electrophoresis inspite of it being highly carcinogenic?
[CBSE Sample Paper 2014]
Ans. Ethidium bromide (EtBr) exchanges its visible range of wavelength with the invisible wavelength
of DNA, to make it visible under UV light.
Q. 17. Mention the type of host cells suitable for the gene guns to introduce an alien DNA.
[CBSE Delhi 2014] [HOTS]
Ans. Plant cells
Q. 18. Why is it not possible for an alien DNA to become part of a chromosome anywhere along its
length and replicate normally? [CBSE (AI) 2014] [HOTS]
Ans. Alien DNA must be linked to ori or origin of replication site to start replication.
Q. 19.Name the host cells in which micro-injection technique is used to introduce an alien DNA.
[CBSE (F) 2014] [HOTS]
Ans. Animal cells.
Q. 20. Write the name of the enzymes that are used for isolation of DNA from bacterial and fungal
cells respectively for Recombinant DNA technology.
[CBSE Delhi 2011; (AI) 2014; (F) 2014 ] [HOTS]
Ans. Bacterial cell is treated with enzyme lysozyme.
Fungal cell is treated with chitinase.
Q. 21. Which main technique and instrument is used to isolate DNA from any plant cell?
[CBSE Sample Paper 2014]
Ans. Centrifugation and centrifuge
Q. 22. What is the cell that receives a recombinant gene called? [CBSE 2019 (57/4/1)]
Ans. Competent host cell/recipient cell.
Q. 23. Why is a thermostable DNA polymerase needed in amplification (genetic engineering)? [HOTS]
Ans. Because thermostable DNA polymerase remains active even at high temperature required for
extension step of PCR.
Q. 24. Identify the reason for selection of DNA polymerase from Thermus aquaticus for Polymerase
Chain Reaction. [CBSE Sample Paper 2016]
Ans. DNA polymerase from Thermus aquaticus remains active during the high temperature induced
denaturation of double stranded DNA.
Q. 25. How is repetitive/satellite DNA separated from bulk genomic DNA for various genetic
experiments? [CBSE Delhi 2014]
Ans. By density gradient centrifugation.
Q. 26. How can bacterial DNA be released from the bacterial cell for biotechnology experiments?
[CBSE Delhi 2011]
Ans. The bacterial cell wall is digested by the enzyme lysozyme to release DNA from the cell.

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Short Answer Questions [2 marks]
Q. 1. Explain the work carried out by Cohen and Boyer that contributed immensely in biotechnology.
[CBSE Delhi 2012]
Ans. Stanley Cohen and Herbert Boyer in 1972 constructed the first recombinant DNA. They isolated
the antibiotic resistance gene by cutting out a piece of DNA from the plasmid of a bacterium
which was responsible for conferring antibiotic resistance. The cut piece of DNA was then linked
with the plasmid DNA of Salmonella typhimurium and transferred to E. coli for transformation.
Q. 2. Explain with the help of an example the relationship between restriction endonuclease and a
palindromic nucleotide sequence. [CBSE (F) 2016]
Ans. Restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA.
Restriction endonuclease cuts the strand of DNA a little away from the centre of palindromic
nucleotide sequence but between the same two bases on the opposite strands, leaving single
stranded portions at the end called sticky ends.

Q. 3. Explain palindromic nucleotide sequence with the help of a suitable example.

[CBSE (F) 2014]
Ans. The palindrome in DNA is a sequence of base pairs that reads same on the two strands when
orientation of reading is kept the same. For example, the following sequences reads the same on
the two strands in 5′ 3′ direction. This is also true if it is read in the 3′ 5′ direction.
5′ — — GAATTC — — 3′
3′ — — CTTAAG — — 5′
Q. 4. Why are molecular scissors so called? Write their use in biotechnology. [CBSE (F) 2014]
Ans. (a) The restriction endonucleases are called molecular scissors, as they cut the DNA segments at
particular locations, e.g., EcoRI.
(b) The restriction enzymes cut the DNA strands a little away from the centre of the palindromic
sites, but between the same two bases on the opposite strands. This leaves single stranded
portions with overhanging stretches called sticky ends on each strand as they form hydrogen
bonds with their complementary cut counterparts. This stickiness at the ends facilitates the
action of the enzyme DNA ligase.
Q. 5. Explain the role of the enzyme EcoRI in recombinant DNA technology. [CBSE (F) 2016]
Ans. EcoRI inspects length of DNA and recognises specific palindromic nucleotide sequences. It then
binds with DNA and cuts each of the two strands of double helix at specific points.

Refer to Basic Concepts Point 4 (Mechanism of Action of Endonucleases).
The first endonuclease discovered was HindII.
Q. 6. Write the convention used for naming restriction enzymes. [CBSE (F) 2011]
OR
Explain with the help of a suitable example the naming of a restriction endonuclease.
[CBSE Delhi 2014]
Ans. The convention for naming restriction enzymes is that the first letter to the name comes from the
Genus and the second two letters come from species and third letter indicates the strain of the
prokaryotic cell from which they are isolated e.g., EcoRI comes from Escherichia coli RI, here R
stands for the strain and I follows the order in which the enzyme was isolated.

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 Q. 7. Name the natural source of agarose. Mention one role of agarose in biotechnology.
Ans. The natural source of agarose is sea weed. Agarose is a natural polymer. It is used to develop the
matrix for gel electrophoresis. It helps in the separation of DNA fragments based on their size.
Q. 8. Write any four ways used to introduce a desired DNA segment into a bacterial cell in
recombinant technology experiments. [CBSE (AI) 2013]
Ans. (i) The desired DNA segment is inserted into a cloning vector and the bacterial cell can be made
to take it up after making them competent by treating them with specific concentration of
divalent cations such as calcium.
(ii) Microinjection
(iii) Biolistics
(iv) Disarmed pathogen vector
Q. 9. State how has Agrobacterium tumefaciens been made a useful cloning vector to transfer DNA
to plant cells. [CBSE Delhi 2014]
Ans. Agrobacterium tumifaciens is known to be a natural vector and consists of a pathogenic plasmid.
It is capable of passing its DNA to plants and induce tumour by integrating its DNA with host
genome. The tumour causing gene in the plasmid of this bacteria is replaced by gene of interest
and is now used as a cloning vector to transfer the DNA into plant cells.
Q. 10. What are ‘cloning sites’ in a cloning vector? Explain their role. Name any two such sites in
pBR322. [CBSE (AI) 2014]
Ans. Cloning sites are the recognition sites on plasmid. The restriction enzymes recognise these sites
for cutting and ligation of alien DNA at this place. For example, EcoRI, BamHI.
Q. 11. (a) Mention the difference in the mode of action of exonuclease and endonuclease.
(b) How does restriction endonuclease function? [CBSE Delhi 2013]
Ans. (a) Exonuclease removes nucleotides from the ends of DNA whereas endonuclease cuts at
specific positions within DNA at specific positions.
(b) Restriction endonuclease recognises and cuts specific palindromic nucleotide sequences in
the DNA.
Q. 12. How does a restriction nuclease function? Explain. [CBSE (AI) 2014]
Ans. Restriction nuclease cuts DNA at specific sites. Nucleases are of two types exonuclease and
endonuclease.
Exonuclease cuts DNA at the ends, whereas endonuclease cuts at specific sites within DNA.
Q. 13. Would you like to choose an exonuclease enzyme while producing a recombinant DNA
molecule? [NCERT Exemplar] [HOTS]
Ans. No, as exonuclease acts on the free ends of linear DNA molecule. Therefore, instead of producing DNA
fragments with sticky ends, it will shorten or completely degrade the DNA fragment containing the gene
of interest, and the circular plasmid (vector) will not get cut as it lacks free ends.
Q. 14. You have created a recombinant DNA molecule by ligating a gene to a plasmid vector. By
mistake, your friend adds exonuclease enzyme to the tube containing the recombinant DNA.
How will your experiment get affected as you plan to go for transformation now?
[NCERT Exemplar] [HOTS]
Ans. The experiment will not likely be affected as recombinant DNA molecule is circular and closed,
with no free ends. Hence, it will not be a substrate for exonuclease enzyme which removes
nucleotides from the free ends of DNA.
Q. 15. Restriction enzymes present in the cloning site of a vector should not have more than one
recognition site. Comment. [NCERT Exemplar] [HOTS]
Ans. If the restriction enzymes have more than one recognition site in a vector, then the vector itself
will get fragmented on treatment with the restriction enzyme.

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Q. 16. A plasmid without a selectable marker was chosen as vector for cloning a gene.
Ans. In a gene cloning experiment, first a recombinant DNA molecule is constructed, where the gene
of interest is ligated to the vector and introduced inside the host cell (transformation). Since, not
all the cells get transformed with the recombinant/plasmid DNA, in the absence of selectable
marker, it will be difficult to distinguish between transformants and non-transformants, because
role of selectable marker is in the selection of transformants.
Q. 17. Write the use of the following in biotechnology.
(a) Chilled ethanol (b) Microinjection
(c) Bioreactor (d) Plasmid [HOTS]
Ans. (a) It is added to precipitate the purified DNA to isolate it.
(b) It is used to inject the foreign gene into a host cell, directly.
(c) It is the set up to culture large volumes of transgenic bacteria to get large quantities of the
product protein.
(d) It is the vector to transform a foreign gene.
Q. 18. How can DNA segments, separated by gel electrophoresis, be visualised and isolated?
Ans. The separated DNA molecules are visualised only after staining DNA with ethidium bromide
followed by exposure to UV radiation. They appear as bright orange coloured bands. The
separated bands of DNA (on the gel) are cut from the agarose gel and extracted from the gel
piece. This process is called elution.
Q. 19. Explain the process of gel-electrophoresis technique. [CBSE 2019 (57/2/2)]
Ans. Refer to Basic Concepts Point 5.
Q. 20. Why does the ‘insertional inactivation’ method to detect recombinant DNA is preferred to
‘antibiotic resistance’ procedure? [CBSE (F) 2016, 2017]
Ans. In insertional inactivation method, the presence of a chromogenic substrate gives blue coloured
colonies in absence of an insert. Presence of an insert in the enzyme site does not produce colour.
This is because insertional inactivation of the β-galactosidase has taken place due to the insert.
Antibiotic resistance method requires duplicate plating. It is a cumbersome procedure to perform.
Q. 21. (a) A recombinant vector with a gene of interest inserted within the gene of a-galactosidase
enzyme, is introduced into a bacterium. Explain the method that would help in selection
of recombinant colonies from non-recombinant ones.
(b) Why is this method of selection referred to as “insertional inactivation”?
[CBSE (AI) 2012] [HOTS]
Ans. (a) Bacteria is grown in a medium with chromogenic substrate, blue coloured colonies show no
recombinations and colonies with no blue colour show presence of recombinants.
(b) Gene for the enzyme is inactivated by insertion of foreign DNA.
Q. 22. How is insertional inactivation of an enzyme used as a selectable marker to differentiate
recombinants from non-recombinants? [CBSE (F) 2014] [HOTS]
Ans. When a recombinant DNA is inserted within the coding sequence of an enzyme β-galactosidase,
it results into inactivation of the enzyme. The bacterial colonies having recombinant plasmid,
show no colouration while those without recombinant plasmid show blue colour.
Q. 23. Why and how bacteria can be made ‘competent’? [CBSE Delhi 2013]
Ans. Bacteria are made competent to accept the DNA or plasmid molecules. This is done by treating
them with specific concentration of a divalent cation such as calcium to increase pore size in
cell wall. The cells are then incubated with recombinant DNA on ice, followed by placing them
briefly at 42°C and then putting it back on ice.
Q. 24. A wine maker and a molecular biologist who has developed a recombinant vaccine, both claim
themselves to be biotechnologist. Who in your opinion is right? [HOTS]

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 Ans. Both are right because biotechnology is a very wide area which deals with techniques of using
a ‘natural’ organism (or its parts) as well as genetically modified organism to produce products
and processes useful for mankind. A wine maker employs a strain of yeast to produce wine
by fermentation (a natural phenomenon), while the molecular biologist has cloned gene for the
antigen (that is used as vaccine) in an organism which allows the production of the antigen in
large amount.
Q. 25. For producing a recombinant protein (for therapeutic purpose) in large scale, which vector
would you choose—a low copy number or high-copy number? [NCERT Exemplar] [HOTS]
Ans. High-copy number, because higher the copy number of vector plasmid, higher the copy number of gene
and consequently, protein coded by the gene is produced in high amount.
Q. 26. DNA being hydrophilic cannot pass through the cell membrane of a host cell. Explain how
does recombinant DNA get introduced into the host cell to transform the latter. [HOTS]
Ans. Refer to Basic Concepts Point 7.
Q. 27. A vector is engineered with three features which facilitate its cloning within the host cell. List
the three features and explain each one of them. [CBSE Sample Paper 2014]
Ans. (i) Origin of replication/ori site—From here the replication starts (and any piece of DNA when
linked, can be made to replicate within the host cell).
(ii) At least two Selectable markers—Helpful in identifying and eliminating non-transformants.
(iii) Unique Restriction sites for more than one restriction enzymes—The foreign DNA links to
this region of the plasmid.
Q. 28. How is DNA isolated in purified form from a bacterial cell?
Ans. DNA, a genetic material is isolated in purified form by treating the bacterial cells with the
enzymes such as lysozyme to remove the cell wall. The RNA thus released can be removed by
treating them with ribonuclease and enzyme proteases is added to remove proteins. Finally,
chilled ethanol is added to precipitate the purified DNA.
Q. 29. Name the source of the DNA polymerase used in PCR technique. Mention why it is used.
[CBSE (AI) 2013]
Ans. The source is the bacterium Thermus aquaticus. It is used because it is thermostable and do not
denature at high temperatures.
Q. 30. Explain any two methods of vectorless gene transfer.
Ans. The two methods of vectorless gene transfer are:
(i) Micro-injection: The technique of introducing foreign gene in a target cell by injecting the
DNA, directly into the nucleus, by micro-needle is called micro-injection.
(ii) Electroporation: It is the process in which transient holes are produced in the plasma
membrane of the target cell, to incorporate foreign DNA.
Q. 31. What is meant by gene cloning? [NCERT Exemplar]
Ans. Gene cloning refers to a process in which a gene of interest is ligated to a vector. The recombinant
DNA thus produced is introduced in a host cell by transformation. Each cell gets one DNA
molecule and when the transformed cell grows to a bacterial colony, each cell in the colony has a
copy of the gene.
Q. 32. Why is Agrobacterium tumifaciens a good cloning vector? Explain.
Ans. Agrobacterium tumifaciens is a soil bacterium which causes disease in many dicot plants. It is able
to deliver a piece of DNA known as T-DNA, to transform the normal cells into tumour cells
and direct these tumour cells to produce the chemicals required by the pathogen. The tumour
inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector
which is no more pathogenic to the plants but still deliver genes of interest into a variety of
plants.
Q. 33. What modification is done in the Ti plasmid of Agrobacterium tumefaciens to convert it into a
cloning vector? [NCERT Exemplar]

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 Ans. T-DNA is the only essential part required to make Ti plasmid a cloning vector. The plasmid is
disarmed by deleting the tumour inducing genes in the plasmid so that it becomes an effective
cloning vector and remove it harmful effect.
Q. 34. What does ‘competent’ refer to in competent cells used in transformation? [NCERT Exemplar]
Ans. Competent means bacterial cells which by various methods like treatment with CaCl2 are made
capable of taking up foreign DNA.
Q. 35. Describe the role of CaCl2 in preparation of competent cells. [NCERT Exemplar]
Ans. CaCl2 is known to increase the efficiency of DNA uptake to produce transformed bacterial cells.
The divalent Ca2+ ions supposedly create transient pores in the bacterial cell wall, by which the
entry of foreign DNA is facilitated into the bacterial cells.
Q. 36. What is the significance of adding proteases at the time of isolation of genetic material
(DNA)? [NCERT Exemplar]
Ans. Role of proteases is to degrade the proteins present inside a cell (from which DNA is being
isolated). If the proteins are not removed from DNA preparation then they could interfere with
any downstream treatment of DNA.
Q. 37. Name the source organism that possesses Taq polymerase. What is so special about the function
of this enzyme? [CBSE (AI) 2012]
OR
Name the organism from where the thermostable DNA polymerase is isolated. State its role in
genetic engineering. [CBSE (F) 2011]
Ans. Source organism: Thermus aquaticus
The enzyme can tolerate high temperature and is thus thermostable. It does not get denatured
during PCR at high temperature.
Q. 38. How are recombinant vectors created? Why is only one type of restriction endonuclease
required for creating one recombinant vector? [CBSE (F) 2011]
Ans. The construction of recombinant DNA is done by linking a gene encoding antibiotic resistance
with a native plasmid. These plasmid DNA act as vectors to transfer the piece of DNA attached
to it.
Only one type of restriction endonuclease is required for creating recombinant vector because
when cut by the same enzyme, the resultant DNA fragments have the same sticky ends, which
can be joined together using DNA ligases.
Q. 39. Name the type of bioreactor shown. Write the purpose for which it is used. [CBSE (AI) 2011]

Ans. The given bioreactor is the simple stirred tank bioreactor.

Its purpose is large scale production of recombinant protein or enzymes, using microbial plants/
animals/human cells.
Q. 40. (a) Explain how to find whether an E.coli bacterium has transformed or not when a recombinant
DNA bearing ampicillin resistant gene is transferred into it.

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 (b) What does the ampicillin resistant gene act as in the above case? [CBSE Delhi 2013] [HOTS]
Ans. (a) E.coli bearing transferred recombinant DNA are first grown on ampicillin containing medium
and then transferred on to a medium containing tetracycline. The transformants will grow
only in ampicillin containing medium and not in tetracycline containing medium. The non-
transformants, on the other hand, will grow in both the mediums.
(b) Ampicillin resistant gene acts as a selectable marker and helps in selecting the transformants.
Q. 41. How can the following be made possible for biotechnology experiments?
(a) Isolation of DNA from bacterial cell.
(b) Reintroduction of the recombinant DNA into a bacterial cell. [CBSE (F) 2012] [HOTS]
Ans. (a) By treating cell with lysozyme
(b) Microinjection/gene gun
Q. 42. Write the role of ‘ori’ and ‘restriction’ site in a cloning vector pBR322. [CBSE Delhi 2014]
Ans. ori is the site where replication starts. This site is responsible for controlling the copy number
of linked DNA. If we want to produce many copies of target DNA, we should clone in a vector
whose ori supports high copy number.
Restriction site is the site of ligation of alien/foreign DNA in the vector, in one of the two antibiotic
resistance site or coding sequence of a-galactosidase.
Q. 43. Rearrange the following in the correct sequence to accomplish an important biotechnological
reaction:
(a) In vitro synthesis of copies of DNA of interest
(b) Chemically synthesised oligonucleotides
(c) Enzyme DNA-polymerase
(d) Complementary region of DNA
(e) Genomic DNA template
(f) Nucleotides provided
(g) Primers
(h) Thermostable DNA-polymerase (from Thermus aquaticus)
(i) Denaturation of dsDNA [CBSE (AI) 2015] [HOTS]
Ans. Correct sequence is
i e b/g g/b c/b h/c f d a
Q. 44. Name the source organism from which Ti plasmid is isolated. Explain the use of this plasmid
in biotechnology.
Ans. Ti plasmid is isolated from Agrobacterium tumifaciens.
Ti plasmid of Agrobacterium tumifaciens has been modified into a cloning vector, which is not
pathogenic to plants but still is able to use the mechanisms to deliver genes of interest into plants.
Q. 45. While doing a PCR, ‘denaturation’ step is missed. What will be its effect on the process?
[NCERT Exemplar]
Ans. If denaturation of double-stranded DNA does not take place, then primers will not be able to
anneal to the template, no extension will take place, hence no amplification will occur.
Q. 46. What would happen when one grows a recombinant bacterium in a bioreactor forget to add
antibiotic to the medium in which the recombinant is growing bacterium? [NCERT Exemplar]
Ans. In the absence of antibiotic, there will be no pressure on recombinants to retain the plasmid (containing
the gene of your interest). Since, maintaining a high copy number of plasmids is a metabolic burden to
the microbial cells, it will thus tend to lose the plasmid.
Q. 47. Is there any difference between recombinant DNA and recombinant protein? Support your
answer. [HOTS]
Ans. rDNA is the plasmid vector containing the foreign DNA whereas recombinant protein is the
product of transgenic gene in the host body or cell.

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Q. 48. Where and why do we use Taq polymerase enzyme when it works exactly as DNA polymerase?
[CBSE Sample Paper 2014] [HOTS]
Ans. In PCR, because it is a thermostable DNA polymerase enzyme, is isolated from bacteria Thermus
aquaticus from hot water springs, and it does not get denatured at high temperature which is
required during PCR and works as normal DNA polymerase enzyme (whereas the normal DNA
polymerase gets denatured at high temperature).
Q. 49. Name the most commonly used bioreactor and describe its working. [CBSE Delhi 2018]
Ans. The most commonly used bioreactor is stirred-tank bioreactor.
A stirred-tank bioreactor is usually cylindrical and have a stirrer which mixes the reactor
contents evenly and makes oxygen available throughout the bioreactor. Optimum conditions of
temperature, pH and foam control are provided.

Long Answer Questions–I [3 marks]

Q. 1. List the steps involved in rDNA technology.
Ans. Steps in rDNA technology:
(i) Isolation of DNA.
(ii) Fragmentation of DNA by restriction endonucleases.
(iii) Isolation of the desired DNA fragments.
(iv) Amplification of the gene of interest.
(v) Ligation of the DNA fragment into a vector using DNA ligase.
(vi) Transfer of recombinant DNA into the host organism.
(vii) Culturing the host cell on a suitable medium on a large scale.
(viii) Extraction of the desired product.
(ix) Downstream processing of the products as finished products are ready for marketing.
Q. 2. List the key tools used in recombinant DNA technology. [CBSE Delhi 2011; (F) 2014] [HOTS]
Ans. The key tools used in recombinant DNA technology are:
(i) Restriction enzymes (ii) Polymerase enzyme
(iii) Ligase enzyme (iv) Vectors
(v) Host organism/cell.
Q. 3. Write the steps you would suggest to be undertaken to obtain a foreign-gene-product.
[CBSE Delhi 2017]
Ans. Refer to Basic Concepts Point 8.
Q. 4.
(a) Explain the significance of ‘palindromic nucleotide sequence’ in the formation of
recombinant DNA.
(b) Write the use of restriction endonuclease in the above process. [CBSE (AI) 2017]
Ans. (a) Palindromic nucleotide sequence is the recognition (specific) sequence present both on
the vector and on a desired or alien DNA for the action of the same (specific) restriction
endonuclease to act upon.
(b) (i) Every endonuclease inspects the entire DNA sequence for the palindromic recognition
sequence.
(ii) On finding the palindrome, the endonuclease binds to the DNA.
(iii) It cuts the opposite strands of DNA in the sugar–phosphate backbone; a little away from
the centre of the palindrome sites but between the same bases on both strands.
(iv) This results in the formation of single stranded overhanging stretches at the end of each
strand called sticky ends.
(v) The sticky ends facilitate the action of the enzyme DNA ligase by readily forming
hydrogen bonds with complementary strands.

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 Q. 5. Explain three basic steps to be followed during genetic modification of an organism.
[CBSE (F) 2017]
Ans. The three basic steps are:
(i) Identification of DNA with desirable genes.
(ii) Introduction of the identified DNA into the host.
(iii) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
Q. 6. Explain the action of the restriction endonuclease EcoRI. [CBSE (F) 2010]
Ans. (i) The recognition sequence shows palindrome character in which the sequence of base pairs
read the same on both the DNA strands, i.e., same in 5′ → 3′ or 3′ → 5′ directions, e.g.,
5′ — G A A T T C — 3′
3′ — C T T A A G — 5′
(ii) The restriction endonuclease acts on specified length of a DNA and binds to the DNA at the
recognition sequence.
(iii) It cuts the opposite double helix of DNA in the sugar-phosphate backbones, a little away
from the centre of the palindrome sites.
(iv) There are overhanging stretches called sticky ends on each strand, which form hydrogen
bonds with their complementary cut counterparts. This stickiness of the ends facilitates the
action of the enzyme DNA ligase.
Q. 7. Prepare a flow chart in formation of recombinant DNA by the action of restriction endonuclease
enzyme EcoRI. [CBSE (F) 2015]
Ans. Refer to Fig. 11.1.
Q. 8. A recombinant DNA is formed when sticky ends of vector DNA and foreign DNA join. Explain
how the sticky ends are formed and get joined. [CBSE (AI) 2010]
OR
EcoRI is used to cut a segment of foreign DNA and that of a vector DNA to form a recombinant
DNA. Show with the help of schematic diagrams.
(i) The set of palindromic nucleotide sequence of base pairs the EcoRI will recognise in both
the DNA segments. Mark the site at which EcoRI will act and cut both the segments.
(ii) Sticky ends formed on both the segments where the two DNA segments will join later to
form a recombinant DNA. [CBSE Delhi 2010]
Ans. The vector DNA and foreign DNA are cut by the same restriction enzyme, such as EcoRI, to form
the same kind of sticky ends. Then these sticky ends are joined by the enzyme DNA ligase.
For figure, refer to Fig. 11.1.
Q. 9. Draw a schematic sketch of pBR322 plasmid and label the following in it:
(a) Any two restriction sites.
(b) ori and rop genes.
(c) An antibiotic resistant gene. [CBSE Delhi 2012]
Ans. Refer to Fig. 11.3.
Q. 10. (a) What is EcoRI? What does ‘R’ represent in this?
(b) Explain its action.
Ans. (a) EcoRI is a restriction endonuclease, obtained from an E. coli bacterium.
R represents the name of the strain.
(b) It cuts the DNA between bases G and A on both the strands only when the sequence GAATTC
is present in DNA.
Q. 11. (a) Name the selectable markers in the cloning vector pBR322? Mention the role they play.
(b) Why is the coding sequence of an enzyme β-galactosidase is a preferred selectable marker
in comparison to the ones named above? [CBSE (AI) 2016]

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Ans. (a) Selectable markers are ampR/ampicillin resistance genes and tetR/tetracycline resistance
gene. They help in identifying and eliminating non-transformants/non-recombinants and
selectively permitting the growth of the transformants/recombinants.
(b) This is because it is simpler and less cumbersome. In the presence of chromogenic substrate
recombinants form colourless colonies and non-recombinants form blue in colonies.
Q. 12. (a) Draw the figure of vector pBR322 and label the following:
(i) Origin of replication
(ii) Ampicillin resistance site
(iii) Tetracycline resistance site
(iv) BamH1 restriction site
(b) Identify the significance of origin of replication. [CBSE Sample Paper 2016]
Ans. (a) Refer to Fig. 11.3.
(b) Origin of replication is responsible for controlling the copy number of the DNA sequence
inserted.
(a) In pBR322, foreign DNA has to be introduced in tetR region. From the restriction enzymes
Q. 13.
given below, which one should be used and why?
PvuI, EcoRI, BamHI
(b) Give reasons, why the other two enzymes cannot be used.
[CBSE Sample Paper 2015, 2017, 2018]
Ans. (a) BamHI should be used, as restriction site for this enzyme is present in tetR region.
(b) PvuI will not be used, as restriction site for this enzyme is present in ampR region (not in tetR).
EcoRI will not be used, as restriction site for this enzyme is not present in selectable marker
tetR.
Q. 14. Name and explain the technique used for separating DNA fragments and making them
available for biotechnology experiments. [CBSE (F) 2013, 2015]
OR
How are the DNA fragments separated and isolated for DNA fingerprinting? Explain.
[CBSE (F) 2015]
Ans. Refer to Basic Concepts Point 5.
Q. 15. Why are genes encoding resistance to antibiotics considered useful selectable markers for
E. coli cloning vector? Explain with the help of one example. [HOTS]
Ans. Genes encoding resistance to antibodies are considered useful selectable markers for E. coli
cloning vector.
If a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred
into E. coli cells, the host cells become transformed into ampicillin-resistant cells. If these
transformed cells are spread on agar plates containing ampicillin, only transformants will grow,
and the non-transformed recipient cells will die as they do not contain the gene for ampicillin
resistance. Thus, transformed cells can be selected. The gene for ampicillin resistance, in this case,
is a useful selectable marker.
Q. 16. How does β-galactosidase coding sequence act as a selectable marker? Explain. Why is it a
preferred selectable marker to antibiotic resistance genes? [CBSE (F) 2017; 2019 (57/4/1)]
Ans. When a recombinant DNA is inserted within the coding sequence of the enzyme β-galactosidase,
it results into inactivation of the enzyme. The presence of a chromogenic substrate gives blue
coloured colonies if the plasmid in the bacteria does not have an insert, whereas presence of
insert do not produce any colour.
Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because
it requires simultaneous plating on two plates having different antibiotics. Therefore, selectable
markers are preferred for selection of recombinants.

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Q. 17. Explain the importance of (a) ori, (b) ampR and (c) rop in the E. coli vector shown below:

rop

Ans. (a) ori: Ori 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. It is also responsible for controlling
the copy number of the linked DNA.
(b) ampR: The ligation of alien DNA is carried out at a restriction site present in any antibiotic
resistance gene.
(c) rop: It codes for the proteins involved in the replication of the plasmid.
Q. 18. A B

(a) Mark the positive and negative terminals.
(b) What is the charge carried by DNA molecule and how does it help in its separation?
(c) How the separated DNA fragments are finally isolated?
[CBSE Sample Paper 2015, 2017, 2018] [HOTS]
Ans. (a) Positive terminal – ‘B’
Negative terminal – ‘A’
(b) DNA is negatively charged. Because of its negative charge, DNA moves towards the positive
electrode (anode).
(c) The separated DNA fragments are separated by elution. The separated bands of DNA are cut
out from the agarose gel and extracted from the gel piece.
Q. 19. A plasmid DNA and a linear DNA (both of the same size) have one site for a restriction
endonuclease. When cut and separated on agarose gel electrophoresis, plasmid shows one
DNA band while linear DNA shows two fragments. Explain. [NCERT Exemplar] [HOTS]
Ans. It is because plasmid is a circular DNA molecule. When cut with enzyme, it becomes linear but
does not get fragmented. Whereas, a linear DNA molecule gets cut into two fragments. Hence, a
single DNA band is observed for plasmid while two DNA bands are observed for linear DNA in
agarose gel.
A Site for restriction
B enzyme

(a)
Restriction
enzyme 5 3
A B
3 5

Circular plasmid Linear plasmid

Same
size

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 (b) Site for restriction enzyme

5 3 Linear
A B DNA molecule
3 5
Small Restriction Large
fragment enzyme fragment
5 3 5 3
A C + C B
3 5 3 5
Size of the
fragments are different

Q. 20. A mixture of fragmented DNA was electrophoresed in agarose gel. After staining the gel with
ethidium bromide, no DNA bands were observed. What could be the reason?
[NCERT Exemplar] [HOTS]
Ans. The reasons that could be possible are as follows:
(i) DNA sample that was loaded on the gel may have got contaminated with nuclease (exo- or
endo- or both) and completely degraded.
(ii) Electrodes were put in opposite orientation in the gel assembly, i.e., anode towards the wells
(where DNA sample is loaded). Since DNA molecules are negatively charged, they move
towards anode and hence move out of the gel instead of moving into the matrix of gel.
(iii) Ethidium bromide was not added at all or was not added in sufficient concentration and so
DNA was not visible.
(iv) After staining with Ethidium bromide it was not observed under UV.
Q. 21.
(a) Why must a cell be made ‘competent’ in biotechnology experiments? How does calcium
ion help in doing so?
(b) State the role of ‘biolistic gun’ in biotechnology experiments. [CBSE (AI) 2016]
Ans. (a) A cell must be made competent so that it can take up the hydrophilic DNA from the external
medium. Divalent calcium ions increases the efficiency, of DNA entering the cell through
pores in the cell wall.
(b) Biolistic gun is used to introduce alien DNA into the plant cell by bombarding them with
high velocity microparticles (gold or tungsten coated with DNA).
Q. 22. Explain the process by which a bacterial cell can be made ‘competent’. Why is it essential to
make bacterial cells ‘competent’ in recombinant DNA technology? [CBSE (F) 2010]
OR
How can be a host made competent? Explain the different methods.
Ans. Refer to Basic Concepts Point 7.
Q. 23. What is Ti plasmid? Name the organism where it is found. How does it help in genetic
engineering? [CBSE Delhi 2011]
Ans. An extra-chromosomal DNA which delivers gene of interest into variety of plants and act as
cloning vector is called Ti plasmid. They are present in Agrobacterium tumifaciens. Ti plasmid
vectors are used for genetic transformation in many dicot plants. The tumour inducing (Ti)
plasmid of Agrobacterium tumifaciens has been modified into a cloning vector which is no more
pathogenic to the plants but is still able to use the mechanisms to deliver genes of interest into a
variety of plants.
Q. 24. Expand the following and mention one application of each:
(i) PCR (ii) ELISA [CBSE Delhi 2013]
Ans.
Expansion Application
(i) PCR Polymerase Chain Reaction Amplification of gene of interest/In
forensic study
(ii) ELISA Enzyme Linked Immunosorbent Assay Diagnostic test for AIDS

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Q. 25. Many copies of a specific gene of interest are required to study the detailed sequencing of
bases in it. Name and explain the process that can help in developing large number of copies
of this gene of interest. [CBSE (F) 2015]
Ans. Polymerase Chain reaction (PCR).
For explanation refer to Basic Concepts Point 8(iv).
Q. 26. How is the amplification of a gene sample of interest carried out using Polymerase Chain
Reaction (PCR)? [CBSE (AI) 2012]
OR
Suggest and describe a technique to obtain multiple copies of a gene of interest in vitro.
[CBSE Delhi 2016]
Ans. Refer to Basic Concepts Point 8(iv) and Fig. 11.5.
Q. 27. How can a bioreactor be made to function at optimal state in order to obtain a desired foreign
gene product? Explain. [CBSE (F) 2017]
Ans. A stirred-tank bioreactor is the most commonly used bioreactor. It comes with a curved base
to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen
availability throughout the bioreactor. The bioreactor has an agitator system, an oxygen delivery
system and a foam control system, a temperature control system, pH control system and sampling
port so that volumes of the cultures can be withdrawn periodically.
Q. 28. Describe the roles of heat, primers and the bacterium Thermus aquaticus in the process of
PCR. [CBSE (AI) 2011]
Ans.  Heat denatures or helps in separation of DNA into two strands.
 Primer–Enzyme DNA Polymerase extend the primers using the nucleotides provided in the

reaction and the genomic DNA as template.

 Thermus aquaticus: It is the source of thermostable DNA polymerase or Taq polymerase.

Q. 29. A schematic representation of polymerase chain reaction (PCR) up to the extension stage is
given below. Answer the questions that follow:

(i) Name the process ‘a’. (ii) Identify ‘b’
(iii) Identify ‘c’ and mention its importance in PCR. [CBSE (F) 2010] [HOTS]
Ans. (i) a—Denaturation process
(ii) b—Primers

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 (iii) c—Taq DNA polymerase. Taq polymerase is a thermostable enzyme which remains active
during the high temperature required for extension of DNA.
Q. 30. (a) List the three steps involved in Polymerase Chain Reaction (PCR).
(b) Name the source organism of Taq polymerase. Explain the specific role of this enzyme in
PCR. [CBSE (F) 2014]
Ans. (a) The three steps involved in polymerase chain reaction (PCR):
(i) Denaturation of double stranded DNA (dsDNA) at high temperature.
(ii) Annealing of two sets of primers.
(iii) Extension of primers to form dsDNA by Taq polymerase and deoxynucleotides.
(b) Source organism of Taq polymerase is the bacterium Thermus aquaticus. This enzyme is heat
tolerant and can repeatedly amplify DNA at high temperatures.
Q. 31. Draw a labelled sketch of sparged stirred-tank bioreactor. Write its application. [CBSE Delhi 2015]
Ans. Refer to Fig. 11.6 (b).
Application: Produces larger biomass leading to higher yields of desired protein.
Q. 32. Name two commonly used bioreactors. State the importance of using a bioreactor.
[CBSE Delhi 2013]
Ans. Two commonly used bioreactors are simple stirred tank bioreactor and sparged stirred tank
bioreactor.
A bioreactor is used for
(i) processing large volumes of culture.
(ii) large scale production of recombinant proteins.
(iii) biologically converting raw materials into specific products. (Any two)
Q. 33. “A very small sample of tissue or even a drop of blood can help determine paternity”. Provide
a scientific explanation to substantiate the statement. [CBSE (AI) 2015] [HOTS]
Ans. (i) DNA from all cells of an individual shows the same degree of polymorphism and therefore
becomes a useful identification tool.
(ii) Polymorphs are heritable and the child inherits 50% of the chromosome from each parent.
(iii) With the help of PCR, the small amount of DNA from blood can be amplified and be used in
DNA finger printing to identify the paternity.

Long Answer Questions–II [5 marks]

Q. 1.(a) With the help of diagrams show the different steps in the formation of recombinant DNA
by action of restriction endonuclease enzyme EcoRI.
(b) Name the technique that is used for separating the fragments of DNA cut by restriction
endonucleases. [CBSE (AI) 2011]
Ans. (a) Refer to Fig. 11.1.
(b) Gel electrophoresis is used for separating the fragments of DNA cut by restriction
endonucleases.
Q. 2. Name and describe the technique that helps in separating the DNA fragments formed by the
use of restriction endonuclease. [CBSE (AI) 2014]
Ans. Gel electrophoresis helps in separating DNA fragments.
DNA fragments are negatively charged then they are forced to move towards anode under an
electric field through agarose gel matrix. The fragments separate according to their size through
sieving effect. Hence the smaller fragments move faster and further than the larger ones.
Refer to Basic Concepts Point 5.
Q. 3. (a) Why are engineered vectors preferred by biotechnologists for transferring the desired
genes into another organism?

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 (b) Explain how do “ori”, “selectable markers” and “cloning sites” facilitate cloning into a
vector.
Ans. (a) Engineered vectors are preferred by biotechnologists because they help in easy linking of
foreign DNA and selection of recombinants from non-recombinants.
(b) Refer to Basic Concepts Point 6(i)–(iii).

Q. 4. (i) Describe the characteristics that a cloning vector must possess.
(ii) Why DNA cannot pass through the cell membrane? Explain. How is a bacterial cell made
‘competent’ to take up recombinant DNA from the medium? [CBSE (AI) 2011]
Ans. (i) A cloning vector must have the following characteristics:
(a) ori or origin of replication which can make large number of copies
(b) Selectable marker i.e., genes encoding for an antibiotic resistance or genes encoding for
α-galactosidase.
(c) Recognition site for the restriction enzyme to recognise.
(ii) DNA is a hydrophilic molecule, therefore it cannot pass through the cell membrane.
The bacterial cells can be made competent by treating them with a specific concentration of
a divalent ion like calcium. The cells are then incubated on ice followed by a heat shock by
placing them briefly at 42°C and then putting back on ice.
Q. 5. If a desired gene is identified in an organism for some experiments, explain the process of the
following:
(i) Cutting this desired gene at specific location.
(ii) Synthesis of multiple copies of this desired gene. [CBSE (AI) 2011]
Ans. (i) The desired gene is cut using the enzymes restriction endonucleases. Firstly, the restriction
endonucleases that recognise the palindromic nucleotide sequence of the desired gene is
identified. The endonuclease inspects the entire DNA sequences to find and recognise the
site. It cuts each of the double helix at a specific point which is a little away from the centre of
the palindromic site. The cutting site is between the same two bases on the opposite strands.
This results in over-hanging single stranded stretches which act as sticky ends.
(ii) Multiple copies of the desired gene is synthesised by polymerase chain reaction (PCR)
method. In this method, the desired gene is synthesised in vitro. The double stranded DNA
is denatured using high temperature of 95°C and the strands are separated. Each separated
strand acts as template.
Two sets of oligonucleotide primers are annealed to the denatured DNA strands. The
thermostable Taq polymerase extends the primers, using nucleotides provided in the reaction
mixture. Finally the amplified fragments are ligated into recipient cells.
Q. 6. (a) Mention the role of vectors in recombinant DNA technology. Give any two examples.
(b) With the help of diagrammatic representation only, show the steps of recombinant DNA
technology.
Ans. (a) Role of vectors: The vectors have the ability to replicate within the bacterial cells independent
of the control of chromosomal DNA. If an alien piece of DNA is linked to the vector like
bacteriophage or plasmid DNA, it can be made to multiply, its number being equal to the
copy number of the vector. Vectors are also used in the selection of recombinants from non-
recombinants. Plasmids and bacteriophages are the most commonly used vectors.
(b) Refer to Fig. 11.4.
Q. 7. Which methodology is used while sequencing the total DNA from a cell? Explain it in detail.
Ans. Methodology used: [CBSE Sample Paper 2017]
OO Sequence Annotation – total DNA from a cell is isolated, converted into random fragments of
relatively smaller sizes, and cloned in suitable host using specialized vectors.

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 OO The cloning resulted into amplification of each piece of DNA fragment.
OO The fragments were sequenced using automated DNA sequencers, these sequences are then
arranged based on some overlapping regions (present in them).
OO This requires generation of overlapping fragments (for sequencing).
Specialised computer based programmes were developed, and these sequences were
OO

subsequently annotated and assigned to each chromosome.

Q. 8. For selection of recombinants, insertional inactivation of antibiotic marker has been
superceded by insertional inactivation of a marker gene coding for a chromogenic substrate.
Give reasons. [NCERT Exemplar] [HOTS]
Ans. Selection of recombinants due to inactivation of antibiotics is a laborious process as it requires:
(i) a vector with two antibiotic resistance markers,
(ii) preparation of two kinds of media plates, with one antibiotic each.
Transformed cells are first plated on the antibiotic plate which has not been insertionally
inactivated (say, ampicillin) and incubated overnight for growth of transformants. For selection
of recombinants, these transformants are replica-plated on second antibiotic (say, tetracycline)
plate (which got inactivated due to insertion of gene). Non-recombinants grow on both the plates
(one carrying ampicillin and the other carrying tetracycline) while recombinants will grow only
on ampicillin plate.
This entire exercise is laborious and takes more time (two overnight incubation) as well. However,
if we choose insertional inactivation of a marker that produces colour in the presence of a
chromogenic compound, we can distinguish between the recombinants and non-recombinants
on a single medium plate (containing one antibiotic and the chromogenic compound) after
overnight growth.
Q. 9.
(a) Explain how recombinants and non-recombinants are differentiated on the basis of colour
production in the presence of a chromogenic substrate. Name that procedure.
(b) Describe the temperature treatment that enhances the bacteria to take up the rDNA.
[HOTS]
Ans. (a) The procedure is called insertional inactivation.
In this method recombinants and non-recombinants are differentiated on the basis of the
ability to produce colour in the presence of a chromosomic substrate. In this method, a rDNA
is inserted in an enzyme – b-galactosidase which leads to inactivation of the enzyme which
does not produce colour due to insertion.
(b) (i) Host cells are incubated with rDNA on ice.
(ii) Followed by placing them briefly at 42°C.
(iii) Then transfer them back on ice.
This enables the host cells (bacteria) to take up the rDNA.

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Self-Assessment Test
Time allowed: 1 Hour Max. marks: 30

1. Choose and write the correct option in the following questions. (3×1 = 3)
(i) Rising of dough is due to
(a) multiplication of yeast (b) production of CO2
(c) emulsification (d) hydrolysis of wheat flour starch into sugars.
(ii) ‘Restriction’ in Restriction enzyme refers to
(a) cleaving of phosphodiester bond in DNA by the enzyme
(b) cutting of DNA at specific position only
(c) prevention of the multiplication of bacteriophage by the host bacteria
(d) all of the above
(iii) Which of the following should be chosen for best yield if one were to produce a recombinant
protein in large amounts?
(a) Laboratory flask of largest capacity
(b) A stirred-tank bioreactor without in-lets and out-lets
(c) A continuous culture system
(d) Any of the above
2. In the following questions a statement of assertion followed by a statement of reason is given.
Choose the correct answer out of the following choices. (3×1 = 3)
(a) Assertion and reason both are correct statements and reason is correct explanation for assertion.
(b) Assertion and reason both are correct statements but reason is not correct explanation for
assertion.
(c) Assertion is correct statement but reason is wrong statement.
(d) Assertion is wrong statement but reason is correct statement.

(i) Assertion : In GMOs desirable DNA segment is introduced into a suitable host.
Reason : The gene transfer is done using a biolistic gun only.
(ii) Assertion : The palindromic sequences at which endonucleases act vary from organism to
organism.
Reason : The palindromic sequence of Eco RI is GAATTC.
(iii) Assertion : A temperature control system is an important requirement for bioreactor.
Reason : Every microorganism or enzyme is functional only at an optimum temperature
conditions.
3. What is plasmid? (1)
4. Why is Taq polymerase preferred in PCR? (1)
5. (a) Why are restriction endonucleases known as molecular scissors?
(b) Give the palindromic sequence recognised by Eco RI. (2)
6. Differentiate between rDNA and cDNA. (2)
7. Which is the most commonly used bioreactor? Explain its functioning. (2)

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 8. Draw a schematic diagram of the E.coli cloning vector pBR322 and mark the following in it :
(a) ori (b) rop
(c) ampicillin resistance gene (d) tetracycline resistance gene (2)
9. Name and explain the techniques used in the separation and isolation of DNA fragments to be
used in recombinant DNA technology. (3)
10. Study the diagram given below and answer the questions that follow: (3×1 = 3)

(i) Why have DNA fragments in band ‘D’ moved farther away in comparison to those in band
‘C’?
(ii) Identify the anode end in the diagram.
(iii) How are these DNA fragments visualised? [CBSE (F) 2011]
11. What are the steps in the process of recombinant DNA technology? (3)
12. (a) Describe the different steps in one complete cycle of PCR.
(b) State the purpose of such an amplified DNA sequence. (5)

Answers
1. (i)—(b), (ii)—(c), (iii)—(c) 2. (i)—(c), (ii)—(c), (iii)—(a)
zzz

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