Biotechnology-principles and

processes
Question 1.
What is the cell that receives a recombinant gene called ? (All India
2019)
Answer:
Host cell is the cell that receives a recombinant gene.
Question 2.
Write the specific point in the palindrome and the bond that is cut by
Eco Rl. (All Indio 2019)
Answer:
Restriction endonuclease Eco RI cuts the DNA at the sequence known
as palindromic sequence, i.e. GAATTC. The type of bond broken by
Eco RI is phosphodiester bond between the G and A bases of the
palindrome. This site is known as restriction site.
Question 3.
Why do DNA fragments move towards the anode during gel
electrophoresis ? (Delhi 2011c)
Answer:
DNA fragments are negatively charged molecules and hence, they
move toward the positive charged anode during gel electrophoresis.
Question 4.
Suggest a technique to a researcher who needs to separate fragments
of DNA. (Delhi 2016)
Or
Mention the use of gel electrophoresis in biotechnology experiments.
(Outside Delhi 2016C)
Answer:
Gel electrophoresis is used to separate the fragments of DNA that
were cut by restriction endonucleases.
Question 5.
Name the technique that is used to alter the chemistry of genetic
material (DNA, RNA) to obtain desired result. (Delhi 2016C)
Answer:
The technique used to alter the chemistry of genetic material to obtain
desired result is called genetic engineering.
Question 6.
Why is it not possible for an alien DNA to become part of a
chromosome anywhere along its length and replicate normally? (All
India 2014)
Answer:
The alien DNA itself cannot multiply and replicate but requires a
specific sequence for initiating its replication called origin of
replication in a chromosome ori acts as the starting point of
replication as they it aids in binding of DNA polymerase.
Question 7.
Mention the type of host cells suitable for the gene guns to introduce
an alien DNA. (Delhi 2014)
Answer:
Plant host cells are suitable for the gene guns to introduce an alien
DNA.
Question 8.
Write the two components of first artificial recombinant DNA
molecule constructed by Cohen and Boyer. (Foreign 2014)
Answer:
The two components of first artificial recombinant DNA molecule
constmcted by Cohen and Boyer are
 Antibiotic resistance gene
 Plasmid of Salmonella typhimurium
Question 9.
Name the host cells in which microinjection technique is used to
introduce an alien DNA. (Foreign 2014)
Answer:
The microinjection technique is usually carried out in animal cell to
inject alien DNA directly into the nucleus.
Question 10.
Name the material used as matrix in gel electrophoresis and mention
its role. (All India 2014C)
Answer:
The material used as matrix in gel electrophoresis is agarose.
This agarose gel acts as a sieve to separate the DNA fragments
according to their size.
Question 11.
Write any four ways used to introduce a desired DNA segment into a
bacterial cell in recombinant technology experiments. (All India
2013)
Answer:
Ways to introduce desired DNA into bacterial cell are
 microinjection
 disarmed pathogen vectors
 treatment of host cell by bivalent cation such as calcium
 biolistic or gene gun
Question 12.
How can retroviruses be used efficiently in biotechnology
experiments inspite of them being disease causing? (All India 2013C)
Answer:
Retroviruses can be used in biotechnology experiments after being
disarmed, i.e. removal of virulent gene so that it is unable to cause
infection in hosts they are transferred to.
Question 13.
State what happens when an alien gene is ligated at Pvu I site of
pBR322 plasmid. (All India 2013C)
Answer:
An alien gene ligated at Pvu I site of pBR322 plasmid cause the
transformant cell to loose the ampicillin-resistance as ampR gene
becomes non-functional. Thus, the recombinant does not grow in the
presence of ampicillin.
Question 14.
Why is ‘plasmid’ an important tool in biotechnology experiments?
(All India 2013C)
Answer:
Plasmid have the ability to replicate within bacterial cells
independently of chromosomal DNA. They have high copy number,
therefore an alien DNA ligated to it, will have equal copy number as
that of plasmids. So, it is used as a vector in gene cloning experiments
and thus acts as an important tool in biotechnology.
Question 15.
State what happens when an alien gene is ligated at Sal I site of
pBR322 plasmid. (Delhi 2013C)
Answer:
When an alien gene is ligated at Sal I site of tetracycline resistance
gene in the vector pBR322, the recombinant plasmid lose its
tetracycline resistance.
Question 16.
Mention the uses of cloning vector in biotechnology. (Delhi 2011)
Answer:
Uses of Cloning Vector in Biotechnology
 Helps in linking the foreign/alien DNA with the host’s DNA.
 Helps in the selection of recombinants from the non-
recombinants.
Question 17.
Biotechnologists refer to Agrobacterium tumefaciens as a natural
genetic engineer of plants. Give reasons to support the statement. (All
India 2011)
Answer:
Agrobacterium tumefaciens is a pathogen of several dicot plants. It is
used as a natural genetic engineer because it is able to deliver a piece
of its DNA (called T-DNA) to transform normal plant cells into
tumour cells. It direct the tumour cells to synthesise the chemicals
required by the pathogen.
Question 18.
Why is it essential to have a selectable marker in a cloning vector?
(All India 2011)
Answer:
Selectable marker in cloning vector helps in identifying and selecting
the recombinants and eliminating the non-recombinants.
Question 19.
In the year’1963, two enzymes responsible for restricting the growth
of bacteriophage in E. coli were isolated. How did enzymes act to
restrict the growth of the bacteriophage? (All India 2011C)
Or
How is the action of exonuclease different from that of endonuclease?
(All India 2010)
Answer:
Two enzymes responsible for restricting the growth of bacteriophage
in E. coli are Exonucleases Remove nucleotides from the ends of
DNA. Endonucleases Cut DNA at specific points.
Question 20.
What is the host called that produces a foreign gene product? What is
this product called? (Foreign 2010)
Answer:
Transgenic organisms or genetically modified organisms are the host
that produces a foreign gene product. Recombinant proteins are the
products formed by these host cells.
Question 21.
ß-galactosides enzyme is considered a better selectable marker. Justify
the statement. (Delhi 2019)
Answer:
Coding sequence of p-galactosidase is a better maker, as the
recombinants and non-recombinants are differentiated on the basis of
their ability to produce colour in the presence of a chromogenic
substrate, while the selection of recombinants due to inactivation of
antibiotic resistant gene is a tedieus and time taking process to grow
them simultaneously on two antibiotics containing media.
Question 22.
Explain the mode of action of Eco RI. (Delhi 2016C)
Or
What is Eco RI? How does Eco RI differ from an exonuclease? (Delhi
2015C)
Or
How does a restriction nuclease function? Explain. (All India 2014)
Answer:
Restriction nucleases function by inspecting the length of DNA
sequence and then binding to specific recognition sequences and
cutting the strands at sugar phosphate backbones.
These nucleases are of two types depending on their mode of action
 Restriction exonucleases cut sequences at terminal ends of
DNA.
 Restriction endonucleases, e.g. Eco RI, cut between the two
bases of recognition sequence.
Question 23.
Write the role of ori and restriction site in a cloning vector pBR322.
(Delhi 2014)
Answer:
Ori is a sequence of DNA from where replication starts. Any piece of
DNA that needs to replicate in the host cell has to be linked to it.
Restriction site is the recognition site made of palindromic sequence
for restriction enzymes.
Question 24.
State how was Agrobacterium tumefaciens been made as a useful
cloning vector to transfer DNA to plant cells. (Delhi 2014)
Answer:
Agrobacterium infects plant tissues by transferring its plasmid T-DNA
to the plant genome. This property of Agrobacterium was exploited to
transfer desired gene to a particular plant. The desired gene is inserted
in the plasmid T-DNA and the engineered Agrobacterium is allowed
to infect that particular plant. Hence, it acts as a natural cloning
vector.
Question 25.
Explain with the help of a suitable example the naming of a restriction
endonuclease. (Delhi 2014)
Answer:
Naming of restriction endonuclease involves the following rules
 The first letter of the enzyme comes from the genus and next
two letters from species of the prokaryotic cell from where
enzymes are extracted.
  The Roman numbers following the name shows the order in
which the enzymes were isolated from the bacterial strain. For
example, Eco RI is derived from Escherichia coli RY 13, Hind II
from Haemophilus influenzae Rd, etc.
Question 26.
How are sticky ends formed on a DNA strand? Why are they called
so? (Delhi 2014)
Answer:
Sticky ends on DNA are formed by the action of enzymes restriction
endonucleases. These enzymes cut the strand of DNA a little away
from the centre of the palindrome sequence between the same two
bases on both the strands.
This results in single-stranded stretches on both the complementary
strands at their ends. These overhanging stretches are called sticky
ends as they form hydrogen bonds with the complementary base pair
sequences.
Question 27.
How is insertional inactivation of an enzyme used as a Selectable
marker to differentiate recombinants from non-recombinants?
(Foreign 2014)
Answer:
The insertional activation of an enzyme, e.g. ß-galactosidase occurs
by inserting the desired gene in the coding region of enzyme. It results
in inactivation of ß-galactosidase gene in recombinants. Due to this,
the recombinant or transformed hosts are unable to produce any
colour when grown on chromogenic substrate. Thus, ß-galactosidase
acts as a selectable marker to differentiate recombinants from non-
recombinants.
Question 28.
Explain palindromic nucleotide sequence with the help of a suitable
example. (Foreign 2014)
Answer:
The palindromic nucleotide sequence is the sequence of base pairs in
DNA that reads the same on both the complementary strands of DNA,
with same orientation of reading.
For example,
5-GAATTC-3′
3-CTTAAG-5′
Question 29.
Why are molecular scissors called so? Write their use in
biotechnology. (Foreign 2014)
Answer:
Molecular scissors are so called because they cut DNA at specific
sequences between base pairs. Molecular scissors or restriction
enzymes cut DNA at desired sequences and generate sticky ends that
facilitate the cut DNA to join with host genome or vector DNA. They
play an important role in genetic engineering or biotechnology. It is
because with the help of these enzymes we can cut the desired gene
and introduce into vectors for expression.
Question 30.
Why is making cells competent essential for biotechnology
experiments? List any two ways by which this can be achieved.
(Delhi 2014C)
Or
Why and how bacteria can be made ‘competent’? Delhi 2013
Answer:
Since, DNA molecules are hydrophilic, they cannot pass through cell
membranes. For recombinant DNA to be integrated into vector or host
genome, it is necessary for the DNA to be inserted in the cell.
Therefore, making the host cells competent is necessary in
biotechnology experiments.
The two ways by which cells can be made competent to take up DNA
are
 Chemical action The host cell is treated with a specific
concentration of divalent cation, i.e. calcium increases the pore
size in the cell membrane. DNA is then incubated with treated
bacterial cell at 42°C, thereby increasing the efficiency of DNA
to enter it through pores in cell wall.
 Heat shock treatment Incubating the cells with recombinant
DNA on ice, followed by brief treatment of heat at 42°C and
again putting them back on ice.
Question 31.
How is an exonuclease functionally different from an endonuclease?
Give an example of any two endonucleases other than Sal I. (Delhi
2013C)
Answer:
Exonucleases are the enzymes which cleave base pairs of DNA at
their terminal ends and act on single-strand of DNA or gaps in doublet
stranded DNA. While, endonuclease cleaves DNA at any point except
the terminal ends and can make cut on one strand or on both the
strands of double-stranded DNA, e.g. Eco R1 and Hind II.
Question 32.
Explain the work carried out by Cohen and Boyer that contributed
immensely in biotechnology. (Delhi 2012)
Answer:
Stanley Cohen and Herbert Boyer constructed the first artificial
recombinant DNA (rDNA) molecule.
They isolated the antibiotic-resistance gene by cutting out a piece of
DNA from a plasmid with the help of restriction enzyme and linked it
to a native plasmid of Salmonella typhimurium with the help of DNA
ligase.
Question 33.
(i) A recombinant vector with a gene of interest inserted within the
gene of α-galactosidase enzyme is introduced into a bacterium.
Explain the method that would help in selection of recombinant
colonies from non-recombinant colonies.
(ii) Why is this method of selection referred to as insertional
inactivation? (All India 2012)
Answer:
(i) The recombinant colonies can be differentiated from non-
recombinant colonies by their inability to produce colour in the
presence of a chromogenic substrate.
The recombinants do not produce any colour, while the non-
recombinants produce a blue colour with chromogenic substrate in the
medium. It occurs because of the presence of α-galactosidase in
former and its absence in latter cells.
(ii) The enzyme α-galactosidase becomes inactivated on insertion of
recombinant DNA, within the coding sequence of enzyme. Thus, the
method is called insertional inactivation.
Question 34.
State the role of UV-light and ethidium bromide during gel
electrophoresis of DNA fragments. (Delhi 2012c)
Answer:
DNA fragments are observed only after staining with ethidium
bromide followed by their exposure to UV radiation. This gives bright
orange colour to DNA fragments.
Question 35.
Explain giving reasons why an alien piece of DNA needs to be
integrated to a specific sequence of host DNA for its cloning. (All
India 2011)
Answer:
Refer to Answer No. 6
Question 36.
List the key tools used in recombinant DNA technology. (Delhi 2011)
Answer:
Key tools used in recombinant technology are restriction enzymes,
polymerases, ligases, cloning vectors and competent host organism or
cells.
Question 37.
Explain the role of Ti plasmids in biotechnology. (Delhi 2011)
Answer:
The Ti-plasmid isolated from Agrobacterium is responsible for the
natural transformation of plant cells into tumours. So, it is modified
into a non-pathogenic vector but still is able to deliver the DNA.
This disarmed plasmid of Agrobacterium is used as a vector for the
transformation of plant cells, thus plays an important role in
biotechnology.
Question 38.
How are recombinant vectors created? Why is only one type of
restriction endonuclease required for creating one recombinant
vector? (Foreign 2011)
Answer:
Creation of recombinant vectors Vector DNA is cut at a particular
restriction site by a restriction enzyme. The alien DNA is then linked
with the vector DNA using enzyme ligase to form the recombinant
vector.
A restriction enzyme recognises and cuts the DNA at a particular
sequence called recognition site. The same restriction enzyme is used
for cutting the DNA segment from both the vector and the other
source, so as to produce same sticky ends in both DNA molecules to
facilitate their joining.
Question 39.
Study the diagram given below and answer the following questions.

(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? (Foreign 2011)
Answer:
(i) In band D, DNA fragments are smaller than those on band C. The
fragments separate according to their size through the sieving effect
provided by the gel. So, the smaller fragments move farther away than
the larger ones.
(ii) B is anode end in the diagram as DNA fragments are moving
towards this end.
(iii) Gel containing DNA fragments are stained with ethidium
bromide and exposed to UV radiation. Orange colour bands of DNA
become visible.
Question 40.
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? (All India 2010)
Answer:
Refer to Answer No. 26.
The sticky ends are joined via complementary factor of two
polynucleotide strands bases and by the action of enzyme, DNA
ligase.
Question 41.
Explain the action of the restriction endonuclease Eco RI. Foreign
2010
Or
(i) Illustrate the recognition sequence of Eco RI and mention what
such sequences are called?
(ii) How does restriction endonuclease act on a DNA molecule? (All
india 2010c)
Answer:
Restriction endonuclease Eco RI cuts the DNA strands a little away
from the centre of the palindromic sequence, but between the same
two bases on the two strands, i.e. G and A, on both the strands. The
site of action of enzyme in palindrome sequence GAATTC is called
recognition site.
(i) Due to this, single-stranded portions called sticky ends, overhang
at the end of each strand.
(ii) Because of the stickiness, they easily form hydrogen bonds with
their complementary counterparts.
Question 42.
How are the DNA fragments separated by gel electrophoresis
visualised and separated for use in constructing recombinant DNA?
(Foreign 2010)
Answer:
The separated DNA fragments by gel electrophoresis are stained with
ethidium bromide.
 By the exposure to UV radiation, the separated DNA fragments
become visible as orange- coloured bands.
 The separated bands of DNA are cut out from the agarose gel
and DNA is extracted from these gel pieces and this process is
called elution.
Question 43.
Expand ‘BAC’ and ‘YAC’. What are they and what is the purpose for
which they are used? (All India 2019)
Answer:
‘BAC’ stands for Bacterial Artificial Chromosome and ‘YAC’ stands
for Yeast Artificial Chromosome. These are vectors used in cloning
DNA. For sequencing the total DNA from cell, the DNA is isolated
and converted into relatively smaller size as fragments. DNA
fragments are cloned in suitable host using specialized vectors, such
as BAC and YAC. Fragments of DNA are then sequenced by
automated DNA sequences.
Question 44.
Explain how ‘sticky ends’ are obtained in a DNA segment. Write their
importance in DNA technology year. (All India 2019)
Answer:
Refer to Answer No. 26.
Role of the sticky ends The sticky ends are produced from hydrogen
bonds with their complementary cut counterparts. The stickiness of
the ends facilitates the action of the enzyme DNA ligase which helps
to region the cut DNA.
Question 45.
(i) Mention the importance of gel-electrophoresis in biotechnology.
(ii) Explain the process of this technique. (All India 2019)
Answer:
(i) DNA fragments formed by the use of restriction endonucleases are
separated by gel-electrophoresis.
(ii) DNA fragments are negatively charged molecules. Thus, they
move towards the positive charged anode under electric field through
the medium. The smaller fragments move farther in the gel as
compared to larger fragments due to the sieving effect of gel.
Question 46.
How does p-galactosidase coding sequence act as a selectable
marker? Why is it a preferred selectable marker to antibiotic
resistance genes? Explain. (All India 2019)
Answer:
Selectable marker It helps in indentifying or selecting transformants
and eliminating non-transformants and selectively permit the growth
of the transformants.
ß-galactosidase acts as a selectable marker by inducing the property of
insertional inactivation in transformed cells. In this process the
recombinants and non-recombinants are differentiated on the basic of
colour production in the presence of chromogenic substrate. A
recombinant DNA is inserted within the coding sequence of an
enzyme ß-galactosidase which results into inactivation of the enzyme.
Therefore, the bacterial colonies having inserted plasmid, show no
colouration while, thoseb without plasmid show blue colour.
Question 47.
Give reason why
(i) DNA cannot pass into a host cell through the cell membrane.
(ii) Proteases are added during isolation of DNA for genetic
engineering.
(iii) Single cloning site is preferred in a vector. (All India 2019)
Answer:
(i) Hydrophobic molecules can diffuse through the lipid bilayer of the
plasma membrane and not hydrophilic molecules. DNA being
hydrophilic with the sugar-phosphate backbone, cannot pass through
the cell membrane.
(ii) Proteases catalyze the breakdown of proteins present in the
solution to its component amino acid. If the proteins are not removed
from DNA preparation then, they could interfere with any
downstream treatment of DNA (such as action of restriction
endonuclease, DNA ligase, etc).
(iii) Single cloning sites are preferred as the the cloning of a sequence
at more than one recognition sites within the vector would generate
several fragments leading to complication in gene cloning.
Question 48.
Describe the formation of recombinant DNA by the action of Eco RI.
(Delhi 2019)
Or
Prepare a flow chart information of recombinant DNA by the action
of restriction endonuclease enzyme Eco RI. (Foreign 2015)
Answer:

Role of Eco RI in Recombinant DNA (In detail) Refer to Answer No.

41.
Question 49.
Explain the roles of the following with the help of an example each in
recombinant DNA technology.
(i) Restriction enzymes
(ii) Plasmids 2018
Answer:
(i) The restriction enzymes are known as molecular scissors. These
enzymes belong to a larger group of enzymes called nucleases, which
are of following two types
 Exonucleases Those, who remove nucleotides from the ends of
the DNA (either 5′ or 3′) in one strand of duplex.
 Endonucleases Those, who make cuts at specific position within
the DNA. Each restriction endonuclease functions by
‘inspecting’ the length of a DNA sequence.
Role of Restriction Enzyme in Recombinant DNA Technology The
restriction endonucleases are used to cut plasmid DNA as well as
foreign DNA at desired sites. The foreign DNA is then inserted into
plasmid DNA and the plasmid takes the foreign DNA into the desired
host organism.
Example The first discovered restriction enzyme is Hind II. It was
isolated from Haemophilus influenzae. It always cuts DNA at 5 GT
(Pyrimidine T Or C (Purine A or G) AC3′ and 3’CA (Purine A or G)
(Pyrimidine T or C) TG5′.
It produces DNA segments with blunt ends.

(ii) Plasmids These are extrachromosomal, self-replicating, double-

stranded, closed and circular DNA molecules. It is found only in
bacteria and few yeast cells.
Role of Plasmid in DNA Recombinant Technology These are used as
vectors to carry the desired gene (foreign genes) into the desired host
organisms.
For example, pBR322 is widely used plasmid vector. In its name P
signifies plasmid, B is from Boliver and R is from Rodriguez. Boliver
and Rodriguez were two scientists who developed pBR322 in 1977.
This plasmid has genes for resistance against ampicillin and
tatracycline. They have restriction sites for enzymes like PvuI, Pst I,
Sal I, Bam HI.

Question 50.
Explain the role(s) of the following in biotechnology
(i) Restriction endonuclease
(ii) Gel-electrophoresis
(iii) Selectable markers in pBR322 (Delhi 2017)
Answer:
(i) Restriction endonucleases These are the bacterial enzymes that cut
dsDNA into fragments after recognising and binding to the specific
nucleotide sequences, known as recognition site. These enzymes are
used to form recombinant molecules of DNA, composed of DNA
from different sources.
(ii) Gel-electrophoresis is the technique which allows the separation
and visualisation of fragments of DNA on an agarose gel matrix.
Since, the DNA fragments are negatively charged molecules, they
separate and move towards the anode (+ ve) under the influence of an
electric field. DNA fragments are separated on the basis of their size
through the sieving effect provided by the gel.
(iii) Selectable markers in pBR322 help in identification and selection
of transformants. pBR322, an E. coli cloning vector has two antibiotic
resistance genes, i.e. for ampicillin and tetracycline, which act as
selectable marker. When a foreign DNA is ligated at the site of
tetracycline resistance (tetR) gene in pBR322, the recombinant
plasmid lose tetracycline resistance due to insertional inactivation of
foreign DNA, but can still be selected out from non-recombinants by
placing the transformants on ampicillin containing medium. The
transformants growing on ampicillin containing medium are then
transferred on tetracycline containing medium. The recombinants
grow on ampicillin containing medium but not on tetracycline one
whereas non-recombinants grow on both the media.
Question 51.
(i) Explain the significance of palindromic nucleotide sequences in
the formation of recombinant DNA.
(ii) Write the use of restriction endonuclease in the above process.
(All India 2017)
Answer:
(i) Palindromic nucleotide sequences in the DNA are group of letters
that form the same words when read both forward and backward. For
example, the following sequence reads the same on the two strands in
5′ → 3’direction as well as 3′ → 5’direction.
5’— GAATTC —3′
3’—CTTAAG —5′
Significance These sequences act as recognition sites which are
recognised by specific restriction endonucleases.
(ii) Use of restriction endonuclease During recombinant DNA
formation, these enzymes recognise and make a cut at specific
positions within the DNA and vector. Due to this function, restriction
endonucleases are also called as molecular scissors.
Question 52.
(i) Name the selectable markers in the cloning vector pBR322.
Mention the role they play.
(ii) Why is the coding sequence of an enzyme p-galactosidase a
preferred selectable marker in comparison to the ones named above?
(All India 2016)
Answer:
(i) Selectable markers in cloning vector pBR322 are ampicillin and
tetracycline antibiotic resistance gene. They help in the selection of
transformants and eliminating the non-transformants.
(ii) The selection of recombinants due to inactivation of antibiotics is
a difficult process and requires simultaneous plating on two plates
having different antibiotics. Thus, enzyme p-galactosidase is preferred
as a selectable marker as it allows to differentiate non-recombinants
from recombinants easily by insertional inactivations technique.
Question 53.
(i) Why must a cell be made ‘competent’ in biotechnology
experiments? How does calcium ion help in doing so?
(ii) State the role of ‘biolistic gun’ in biotechnology experiments. (All
India 2016)
Answer:
(i) Refer to Answer No. 30.
(ii) Biolistic guns or gene guns are used to bombared rDNA loaded on
gold or tungston particles with high velocity into host cells. In this
way, the rDNA is delivered to the desired host cells.
Question 54.
How does Agrobacterium tumefaciens act as a suitable vector in the
biotechnological experiments? Site an example where it has been
successfully used as a vector. (Outside Delhi 2016C)
Answer:
Refer to Answer No. 24.
Agrobacterium has been used as vector to introduce a gene from
Tobacco Mosaic Virus (TMV) into tobacco plants.
Question 55.
Describe a palindrome with the help of an example. (Delhi 2016C)
Answer:
Refer to Answer No. 51 (i).
Question 56.
(i) Why was a bacterium used in the first instance of the construction
of an artificial recombinant DNA molecule?
(ii) Name the scientists who accomplished this and how. (Delhi
2016C)
Answer:
(i) A bacterium Salmonella typhimurium was used in the first instance
of construction of artificial recombinant DNA molecule because of
the possibilities of linking a gene encoding antibiotic resistance with a
native plasmid of the bacterium. This was made possible by the
availability of restriction enzymes and the enzyme DNA ligase.
(ii) Stanley Cohen and Herbert Boyer accomplished this in 1972 by
isolating the antibiotic resistance gene by cutting out a piece of DNA
from a plasmid which was responsible for conferring antibiotic
resistance. The cutting of DNA at specific sites was possible with the
availability of restriction enzymes. The cut DNA was linked with the
plasmid DNA using the enzyme DNA ligase. The plasmid DNA acts
as vector to transfer the piece of DNA attached to it.
Question 57.
State the functions of the following in the cloning vector pBR322
(i) ori
(ii) rop and
(iii) Hind III sites (All India 2015C)
Answer:
(i) Refer to Answer No. 23. (ori)
(ii) rop in pBR322 encodes for protein involved in plasmid
replication.
(iii) Hind III is a restriction site in pBR322, where Hind III
endonuclease makes a cut for the introduction of foreign DNA
segment.
Question 58.
Name and explain the technique used for separating DNA fragments
and making them available for biotechnology experiments. (Foreign
2015; All India 2014)
Answer:
DNA fragments formed by the use of restriction endonucleases are
separated by gel electrophoresis.
(i) DNA fragments are negatively charged molecules. Thus, they
move towards the positive charged anode under electric field through
the gel medium.
(ii) DNA fragments separate according to their size due to sieving
effect of agarose gel.
(iii) The separated DNA fragments can be viewed by staining the
DNA with ethidium bromide followed by exposure to UV radiation.
(iv) The separated bands of DNA are cut and extracted from gel piece.
This is known as elution.
Question 59.
Draw a schematic diagram of the E. coli cloning vector pBR322 and
mark the following in it
(i) ori
(ii) rop
(iii) Ampicillin resistance gene
(iv) Tetracycline resistance gene
(v) Restriction site Bam HI
(vi) Restriction site Eco RI (Delhi 2015)
Or
Draw a schematic sketch of pBR322 plasmid and label the following
in it
(i) Any two restriction sites
(ii) ori and rop genes
(iii) An antibiotic resistant gene. (Delhi 2012)
Answer:
E. coll cloning vector pBR 322.
Refer to figure 11.3 on page no. 278.
Question 60.
(i) Draw schematic diagrams of segments of a vector and a foreign
DNA with the sequence of nucleotides recognised by Eco RI.
(ii) Draw the vector DNA segment and foreign DNA segment after the
action of Eco RI and label the sticky ends produced. (Delhi 2014C)
Answer:
(i) Refer to figure 11.1. on page no. 276.
(ii) Refer to figure 11.1 on page no. 276.
Question 61.
What are ‘cloning sites’ in a cloning vector? Explain their role. Name
any two such sites in pBR322. (All India 2014C)
Answer:
The cloning sites contain the specific unique recognition sequence for
a particular restriction enzyme, so as to link the foreign DNA with the
vector DNA and thus, create a recombinant DNA molecule.
These sites are important for joining the DNA fragments of vector and
alien DNA. Multiple recognition sequences for a particular restriction
enzyme within a DNA or vector complicate the process of gene
cloning. The two cloning sites in pBR322 are Bam HI for tetracycline
resistant gene and Pvu I for ampicillin resistant genes.
Question 62.
(i) Explain the basis on which the gel electrophoresis technique
works.
(ii) Write any two ways the products obtained through this technique
can be utilised. (Delhi 2013C)
Answer:
(i) Refer to Answer No. 50 (ii).
(ii) Products obtained via gel electrophoresis can be utilised in
following ways
(a) To construct a recombinant DNA molecule by joining them with
cloning vector.
(b) For amplification of desired segment using Polymerase Chain
Reaction (PCR).
Question 63.
How are the following used in biotechnology ?
(i) Plasmid DNA
(ii) Recognition sequence
(iii) Gel electrophoresis (All India 2011c)
Answer:
(i) Plasmid DNA It is used for constructing recombinant DNA, by
ligating the alien piece of DNA with it. It is used as a cloning vector
and helps in the selection of recombinants from non-recombinants.
(ii) Recognition sequences These are specific base sequences of DNA,
where restriction enzyme cuts the DNA. They are utilised to extract
the desired gene or fragments from DNA molecules.
(iii) Gel electrophoresis It is a technique, used to separate the DNA
fragments according to their size through seiviirg effect of the gel.
Question 64.
(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. (All India 2010)

Answer:
(i) Escherichia coli(E.coli)
(ii) Ori in the vector is responsible for controlling the copy number of
inserted gene.
(iii) The genes encoding resistance to antibiotics like tetR resistant to
tetracycline and ampR resistant to ampicillin are used as selectable
markers. If a foreign DN A is ligated at the Bam HI site of tetracycline
resistance gene, the recombinant plasmids will lose the tetracycline
resistance due to insertional inactivation but transformants can be
selected by growing them on ampicillin containing medium. The
selectable markers help in identifying and eliminating non-
transformants and selectively permitting the growth of transformants.
Question 65.
(i) Eco RI is a restriction endonuclease.
How is it named so? Explain.
(ii) Write the sequence of DNA bases that the enzyme recognises.
Mention the point at which the enzyme makes a cut in the DNA
segment. (Delhi 2010C)
Answer:
(i) Refer to Answer No. 25.
(ii) Refer to Answer No. 41.
Question 66.
(i) Name the technique used for separation of DNA fragments.
(ii) Write the type of matrix used in this technique.
(iii) How is the separated DNA visualised and extracted for use in
recombinant technology? (All India 2010)
Answer:
(i) Gel electrophoresis.
(ii) Refer to Answer No. 10.
(iii) Refer to Answer No. 42.
Question 67.
Unless the vector and source DNA are cut, fragments separated and
joined, the desired recombinant vector molecule cannot be created.
(i) How are the desirable DNA sequences cut?
(ii) Explain the technique used to separate the cut fragments.
(iii) How are the resultant fragments joined to the vector DNA
molecule? (Delhi 2015C)
Answer:
(i) The desirable DNA sequences are cut by using resriction
endonuclease enzyme. These enzymes cut at specific site in
palindromic sequence between same two bases on both the strands.
(ii) Refer to Answer No. 58.
(iii) The resulting fragments are joined together with vector DNA by
DNA ligase enzyme. It forms phosphodiester bonds between them.
Question 68.
(i) Describe the characteristics 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? (All India 2011)
Answer:
(i) Features which facilitate cloning of vector are:
(a) Origin of replication (ori)
 This is the sequence of DNA from where replication starts.
 Any piece of alien or foreign DNA linked to it is made to
replicate within host cell. It also determine the copy number of
the linked DNA.
(b) Selectable jmarker is a marker gene, which helps in selecting the
transformants or recombinants from the non-recombinant ones, e.g.
ampicillin and tetracycline resistant genes in E. coli.
(c) Cloning site is a unique recognition site in a vector to link the
foreign DNA. The presence of a particular cloning or recognition site
helps the particular restriction enzyme to cut the vector DNA.
(d) Small size of the vector The small size facilitates the introduction
of the DNA into the host easily.
(ii) Refer to Answer No. 30.
Question 69.
(i) With the help of diagrams show the different steps in the formation
of recombinant DNA by action of restriction endonuclease enzyme
Eco RI.
(ii) Name the technique that is used for separating the fragments of
DNA cut by restriction endonucleases. (All India 2011)
Answer:
(i) Refer to figure 11.1 on page no. 276.
(ii) Refer to Answer No. 58.
Question 70.
Name the enzymes that are used for the isolation of DNA from
bacterial and fungal cells for recombinant DNA technology. (All India
2014; Foreign 2014)
Answer:
The enzymes used for the isolation of DNA from bacterial cells is
lysozyme and those from fungal cells is chitinase.
Question 71.
How can bacterial DNA be released from the bacterial cell for
biotechnology experiments? (Delhi 2o11)
Answer:
Bacterial cells are treated with lysozyme which digest their cell wall
for releasing DNA.
Question 72.
Why is the enzyme cellulase used for isolating genetic material from
plant cells hut not for animal cells? (Delhi 2010)
Answer:
Cellulase is used for digesting the cellulosic cell wall of plant cells.
Animal cells do not contain cell wall, so cellulase is not required.
Question 73.
How is a continuous culture system maintained in bioreactors and
why? (Delhi 2019)
Answer:
The cells can be multiplied in a continuous culture system. In this, the
used medium is drained out from one side, while fresh medium is
added from the other side to maintain the cells in their physiologically
most active (log/exponential) phase. This type of culturing method
produces a larger biomass leading to higher yields of desired
products.
Question 74.
Name the source of the DNA polymerase used in PCR technique.
Mention why is it used? (All India 2013, 2012; Foreign 2011)
Answer:
The DNA polymerase used in PCR is Taq polymerase which is
extracted from Thermus aquaticus. It is a thermostable enzyme that
can withstand high temperature used in the denaturation and step for
the separation of DNA strands. Hence, it can be used for a number of
cycles of DNA amplification without being denatured.
Question 75.
Name the type of bioreactor shown. Write the purpose for which it is
used. (All India 2011)
Answer:
In the given figure, simple stirred-tank bioreactor is shown.
Bioreactors are used to produce large quantities of the desired gene
products.
Question 76.
What is genetic engineering? List the steps in rDNA technology. (All
India 2011)
Answer:
Genetic engineering is the process of artificial synthesis, isolation,
modification, combination, addition and repair of genetic material as
needed.
Steps of rDNA technology involve
 Isolation of genetic material
 Cutting of DNA at specific locations
 Amplification of gene of interest using PCR
 Preparation and insertion of rDNA into host cell
 Obtaining desirable gene product.
Question 77.
(i) Mention the number of primers required in each cycle of
Polymerase Chain Reaction (PCR). Write the role of primers and
DNA polymerase in PCR.
(ii) Give the characteristic feature and source organism of the DNA
polymerase used in PCR. (All India 2010)
Answer:
(i) Two sets of primers are required in each cycle of polymerase chain
reaction. Primers are required for the addition of nucleotides to make
multiple copies of the DNA of interest. The enzyme DNA polymerase
extends the primers by using the nucleotide provided.
(ii) Refer to Answer No. 5.
Question 78.
A schematic representation of Polymerase Chain Reaction (PCR) upto
the extension stage is given below. Answer the questions that follows.

(i) Name the process A.

(ii) Identify B.
(iii) Identify C and mention its importance in PCR. (Foreign 2010)
Answer:
(i) A-Denaturation of the double-stranded DNA
(ii) B-Primers
(iii) C-DNA polymerase or Taq polymerase
Importance in PCR
It extends the primers using the nucleotides provided in the reaction
medium and the genomic DNA as the template. Taq polymerase is
thermostable enzyme and it can withstand the high temperature used
in denaturation step.
Question 79.
Any recombinant DNA with a desired gene is required in billion
copies for commercial use. How is the amplification done? Explain.
(Delhi 2010C)
Answer:
Amplification of recombinant DNA gene is done using Polymerase
Chain Reaction (PCR).
It is carried out in the following steps
 Denaturation The double-stranded DNA is denatured by
applying high temperature of 95°C for 15 seconds. Each
separated strand acts as a template.
 Annealing Two sets of primers are added, which anneal to the 3′
end of each separated strand.
 Extension Taq polymerase extends the primers by adding
nucleotides complementary to the template provided in the
reaction. Taq polymerase is used in the reaction because it can
tolerate high temperature. All these steps are repeated many
times to get several copies of the desired DNA.
Question 80.
Describe the roles of (i) high temperature, (ii) primers and (iii)
bacterium.
Thermus aquaticus in carrying the process of polymerase chain
reaction. (All India 2019)
Answer:
Role of Heat In PCR (in vitro), the DNA strands are separated by
heating at 95°C for two minutes. Heating causes the breakage of H-
bonds between bases of two strands leading to its unwinding.
Role of Primers Primers are short lengths of DNA of about 20bp long
that are required to start DNA polymerisation in PCR. The primers
hybridise to their complementary sequence on the DNA strands at 40-
50°C temperature and help in DNA polymerisation.
Role of Thermus aquaticus An enzyme called Taq polymerase is
isolated from Thermus aquaticus. Since, this bacteria thrives in
temperature as high as 95°C, this enzyme can also tolerate high
temperature without undergoing denaturation. Therefore, this enzyme
is used in PCR instead of normal DNA polymerase.
Question 81.
Describe the process of amplification of ‘gene of interest’ using PCR
technique. (Delhi 2019)
Answer:
Refer to Answers No. 10
Question 82.
Explain three steps involved in polymerase chain reaction.
(i) List the three steps involved in Polymerase Chain Reaction (PCR).
(ii) Name the source organism of Taq polymerase.
Explain the specific role of this enzyme in PCR. (2018C; Foreign
2014)
Answer:
(i) Refer to Answer No. 10.
(ii) Refer to Answer No. 5.

Question 83.
(i) How has the development of bioreactor helped in biotechnology?
(ii) Name the most commonly used bioreactor and describe its
working. (2018)
Answer:
(i) Bioreactors These are the large volume vessels approximately
(100-1000 L) in which raw materials are biologically converted into
specific products, individual enzymes, etc., using microbial, plant,
animal or human cells. A bioreactor provides the optimal conditions
for achieving the desired product by providing optimum growth
conditions like temperature, pH, substrate, salts, vitamins and oxygen.
The cells can also be multiplied in a continuous culture system.
In this, the used medium is drained out from one side while fresh
medium is added from the other side to maintain the cells in their
physiologically most active log/exponential phase. This type of
culturing method produces a larger biomass leading to higher yields
of desired protein. Thus, it plays a very important role particularly in
traditional biotechnology.
(ii) The most commonly used bioreactor is simple stirred tank
bioreactor. It is usually cylindrical or with a curved base to facilitate
the mixing of the reactor contents. The stirrer activity facilitates even
mixing and availability of 02 throughout the bioreactor. Alternatively
air can also be bubbles into the medium. It has an agitator system, a
temperature control system, a pH control system and sample ports so
that small samples can be withdrawn periodically.
Question 84.
Describe the roles of heat, primers and the bacterium Thermus
aquaticus in the process of PCR. (All India 2017)
Answer:
Refer to Answer No. 11
Question 85.
Write the steps you would suggest to be undertaken to obtain a
foreign-gene product. (Delhi 2017)
Answer:
To obtain a foreign-gene product following steps should be
undertaken
 Identification of DNA with desirable genes.
 Introduction of the identified DNA into suitable host to form
recombinant DNA (rDNA).
 Maintenance of introduced DNA in particular host and gene
cloning.
 Transfer of the DNA (gene transfer) to its progeny.
 Selection of the recombinants from non-recombinants.
 Expression of gene of interest by culturing recombinant cells.
 Culturing of cells in bioreactors for large scale production of
desired gene product. (3)
Question 86.
Suggest and describe a technique to obtain multiple copies of a gene
of interest in vitro. (All India 2016)
Answer:
Polymerase Chain Reaction (PCR) is a technique to obtain multiple
copies of a gene of interest in vitro. This technique amplifies DNA
through a simple enzymatic reaction. This technique was developed
by Kary Mullis in 1965.
The basic requirements of a PCR are the following
 DNA template
  Primers
 Enzyme-Taq polymerase
For detailed description of process, Refer to Answer No. 10.
Question 87.
Why is Taq polymerase preferred in PCR? Mention the source of this
enzyme. (Delhi 2015C)
Answer:
Refer to Answer No. 11.
Question 88.
Draw a labelled sketch of sparged stirred-tank bioreactor. Write its
application. (Delhi 2015)
Answer:
Sparged stirred-tank bioreactor

Application These bioreactors are used to produce large quantities of

products, enzymes, etc., using microbial, plant, animal or human
cells. (1)
Question 89.
Rearrange the following in the correct sequence to accomplish an
important biotechnological reaction
(i) In vitro synthesis of copies of DNA of interest
(ii) Chemically synthesised oligonucleotides
(iii) Enzyme DNA polymerase
(iv) Complementary region of DNA
(v) Genomic DNA template
(vi) Nucleotides provided
(vii) Primers
(viii) Thermostable DNA polymerase (from Thermus aquaticus)
(ix) Denaturation of dsDNA. (All India 2015)
Answer:
The correct sequence of reactions are

Question 90.
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.
(Foreign 2015)
Answer:
PCR is the process that can help in developing large number of copies
of a gene of interest.
Process of PCR Refer to Answer No. 10.
Question 91.
(i) What is a bioreactor? How does it work?
(ii) Name two commonly used bioreactors. (Delhi 2014c)
Answer:
(i) Bioreactors are large vessels in which raw materials are
biologically converted into specific products by microbes, plant and
animal cells or human cells. The bioreactors work by providing
optimal conditions to process the culture as well as the production of
desired product by maintaining optimum pH, temperature, oxygen
and other grpwth conditions required.
(ii) The two commonly used bioreactors are
  Simple stirred-tank bioreactors.
 Sparged stirred-tank bioreactors.
Question 92.
What is a bioreactor used for? Name a commonly used bioreactor and
any two of its components. (All India 2014C)
Answer:
Refer to Answer No. 22.
The components of a commonly used stirred-tank bioreactor are
 Inlet for sterile air or oxygen
 Agitator system
 Temperature control system
 pH control system
 Foam control system
 Sampling ports
Question 93.
Explain in sequence the process of amplification of a gene of interest
using polymerase chain reaction. (All India 2013C, 2012, 2012C)
Or
How is the amplification of a gene sample of interest carried out using
Polymerase Chain Reaction (PCR)? (All India 2012)
Or
Describe the process of gene amplification for rDNA technology
experiments. (All lndnr2011C)
Answer:
Refer to Answer No. 10.
Refer to figure 11.4 on page no. 291.
Question 94.
(i) Describe the different steps in one complete cycle of PCR.
(ii) State the purpose of such an amplified DNA sequence. (AII India
2015C)
Answer:
(i) Refer to text on page no. 291.
(ii) Applications of PCR technique
  In diagnosis of pathogens for specifc infections, like HTV
 To identify mutations in organisms
 In DNA fingerprinting
 Prenatal diagnosis
 Gene therapy

Question 95.
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. (All India 2011)
Answer:
(i) Cutting of desired gene at specific location is done by incubating
the DNA with specific restriction endonuclease. Restriction enzymes
recognise a particular palindromic nucleotide sequence and cuts the
DNA at that site.
(ii) Synthesis of multiple copies of desired gene is carried out by
Polymerase Chain Reaction (PCR).
Refer to Answer No. 10.
Question 96.
Write the function of the following in biotechnology. (Outside Delhi
2016C)
(i) Polymerase chain reaction technique.
(ii) Restriction endonucleases.
(iii) Bacterium Thermus aquaticus.
Answer:
(i) Polymerase chain reaction technique is used to prepare multiple
copies of a gene of interest in vitro using two sets of primers and the
enzyme DNA polymerase.
(ii) Restriction endonucleases are enzymes that make cut at specific
positions within DNA.
(iii) Bacterium Thermus aquaticus produces an enzyme Taq
polymerase which is heat stable, i.e. resistant to denaturation by heat.
The enzyme is used to amplify a specific DNA fragment in PCR
technique.
Question 97.
Explain the basis on which the gel electrophoresis technique works.
Write any two ways the products obtained through this technique can
be utilised. (Delhi 2013C)
Answer:
Gel electrophoresis technique works on the principle of separation of
DNA fragment on the basis of their size and electric charge.
Since, DNA is negatively charged molecule so, they can be separated
according to their size when they towards anode under an electric
field through a medium or matrix (commonly used is agarose).
Shorter molecules move faster towards anode and migrate farther than
the longer one.
The products obtained through this technique can be utilised in the
following ways
 Construction of recombinant DNA by joining with cloning
vectors.
 Used in making multiple copies of same DNA by using PCR
(Polymerase Chain Reaction).
Question 98.
How can the following be made possible for biotechnology
experiments?
(i) Isolation of DNA from bacterial cell.
(ii) Reintroduction of the recombinant DNA into a bacterial cell.
(Foreign 2012)
Answer:
(i) Isolation of DNA from bacterial cell can be done by
 treating the bacterial cells with enzymes such as lysozyme to
remove cell wall.
 RNA associated with DNA can be removed by the treatment
with ribonuclease, whereas protein can be removed by treatment
with protease. Similarly other molecules (if any) are removed by
appropriate treatment.
(ii) Reintroduction of the recombinant DNA into bacterial cell can be
done by the following methods
 The recipient bacterial cell is made ‘competent’ to take up the
recombinant DNA its by the treatment with a specific increased
concentration of calcium ions.
 The recombinant DNA is forced into the cells by heat shock
treatment, i.e. by incubating the cells with rDNA on ice
followed by placing them at 42°C (heat shock) and then again
putting them back on ice. This enables bacteria to take up
rDNA.
Question 99.
(i) Identify A and B illustrations in the following

(i) Write the term given to A and C and why?

(iii) Expand PCR. Mention its importance in biotechnology. (Delhi
2011)
Answer:
(i) (a) A is recognition or restriction site (GAATTC), which is
recognised by restriction enzyme Eco RI.
(b) B is rop gene protein involved in the replication of plasmid coded
by this gene.
(ii) A and C are called palindromes. These are sequence of base pairs
that reads same on the two strands of DNA, when orientation of
reading is kept same.
(iii) PCR is Polymerase Chain Reaction in which multiple copies of
the gene of interest can be synthesised in vitro. Thus, PCR can be
utilised to amplify a single gene or fragment into thousands of copies
to be used in cloning experiments.
Question 100.
Industrial production of biologically important recombinant products
utilises bioreactors. Sohan was aware of this information but was
curious to know about the process involved. He asked his teacher
about this, who explained the process in detail to him.
(i) What are bioreactors? Name the most common type of bioreactor
utilised by industries.
(ii) What is the sequence of events after completion of the
biosynthetic phase in bioreactor ?
Answer:
(i) Bioreactore are large volume vessels in which raw materials are
biologically converted into specific products. The most common type
of bioreactors are of stirring type.
(ii) The sequence of events after the completion of biosynthetic phase
is called as downstream processing. It involves (i) separation and (ii)
purification of products Obtained.
Question 101.
DNA amplification has become an extensively employed technique in
various fields. Arun got curious about this process and asked his
father, a molecular scientist to explain his queries.
(i) What is meant by DNA amplification and how is it achieved?
(ii) Write down the basic steps involved in this process.
(iii) What values do you observe in Arun?
Answer:
(i) DNA amplification refers to the production of multiple copies of
gene of interest from a single copy.
It is achieved by using PCR, i.e. Polymerase Chain Reaction.
(ii) Steps involved in PCR are
 Denaturation of DNA strands at 95°C.
 Annealing with primers at 45°C
 Extension using Taq polymerase and dNTPs.
(iii) Values shown by Arun are inquisitive mind, intelligence and
awareness towards scientific developments