Biotechnology: Principles and Processes

1. Which of the following is responsible for

initiating replication in a plasmid?

a) Gene to be cloned

b) Complete plasmid

c) Origin of replication

Ligase
d)
 Discussion

● Plasmids are autonomously replicating as they

have a specific DNA sequence known as ‘origin
of replication’(ori)
● Any fragment of foreign DNA linked to the ori can
be made to replicate

Origin of
replication

E. coli Plasmid
 1. Which of the following is responsible for
initiating replication in a plasmid?

a) Gene to be cloned

b) Complete plasmid

c) Origin of replication

d) Ligase
 2. Match the following

Column I Column II
A. Vector i. Molecular scissors

B. DNA Ligase ii. Carrier

C. Host iii. Molecular glue

D. Restriction enzyme iv. Bacterial cell

b) A - ii, B - i, C - iii, D - iv
a) A - ii, B - i, C - iv, D - iii

c) A - ii, B - iii, C - iv, D - i d) A - i, B - ii, C - iii, D - iv

 Discussion

Vector - DNA molecule that acts as a vehicle to carry

a desired DNA sequence into a host cell

Host - It can be a plant or animal cell or a microbe.

They carry the recombinant DNA

Genomic DNA Plasmid

E. coli
 Discussion
● Restriction enzymes (Molecular scissors) - Enzymes
used in cutting DNA strands in a particular manner

● Ligases (Molecular glue) - Enzymes that are capable

of catalysing the reaction of joining two DNA fragments
by establishing a phosphodiester bond

Restriction Restriction
enzymes enzymes DNA fragment 1

DNA DNA
ligase ligase

Plasmid DNA DNA fragment 2

 2. Match the following

Column I Column II
A. Vector i. Molecular scissors

B. DNA Ligase ii. Carrier

C. Host iii. Molecular glue

D. Restriction enzyme iv. Bacterial cell

a) A - ii, B - i, C - iv, D - iii b) A - ii, B - i, C - iii, D - iv

c) A - ii, B - iii, C - iv, D - i d) A - i, B - ii, C - iii, D - iv

3. A restriction endonuclease enzyme binds to the
DNA and cuts

a) any one strand of the double helix

each of the two strands at specific points in

b) their base - sugar bonds

each of the two strands at specific points in

c) their base - phosphate bonds

each of the two strands at specific points in

d) their sugar- phosphate backbone
 Discussion

5’

3’ -

Sugar - Phosphate
backbone
Phosphodiester
bond
-

3’

5’ Restriction
endonuclease
3. A restriction endonuclease enzyme binds to the
DNA and cuts

a) any one strand of the double helix

each of the two strands at specific points in

b) their base - sugar bonds

each of the two strands at specific points in

c) their base - phosphate bonds

each of the two strands at specific points in

d) their sugar- phosphate backbone
4. Which of the following statements regarding
EcoR I is not correct?
I. It is isolated from Escherichia coli RY13.
II. Its recognition sequence is
5' GAATTC 3'
3' CTTAAG 5'
III. It produces complementary blunt ends.
IV. It was the first restriction endonuclease to
be characterised.

a) I and II b) II and IV

c) III and IV d) I and IV

 Discussion

● EcoR I is a restriction endonuclease.

● It has been isolated from the bacteria
Escherichia coli RY13.
● It recognises the base sequence 5’ GAATTC 3’
in the double stranded DNA and makes the
cuts between G and A.

5’ G AATTC 3’
3’ C T TAA A GG 5’

Recognition
Sequence EcoRI
 Discussion

● As the cut is made slightly away from the centre, it

results in the formation of DNA fragments with single
stranded overhanging free ends called sticky ends.

Overhang
5’ AATTC 3’
G
3’ CTTAA G 5’

Overhang
 Discussion

Hind II
● The first restriction endonuclease
discovered was Hind II
● This enzyme was first isolated from
Haemophilus influenzae Rd strain II
● The recognition sequence of Hind II
is a series of six base pairs
● It produces blunt ends
4. Which of the following statements regarding
EcoR I is not correct?
I. It is isolated from Escherichia coli RY13.
II. Its recognition sequence is
5' GAATTC 3'
3' CTTAAG 5'
III. It produces complementary blunt ends.
IV. It was the first restriction endonuclease to
be characterised.

a) I and II b) II and IV

c) III and IV d) I and IV

 5. Assertion: A bacterial cell with restriction enzymes
will be easily infected and lysed by bacteriophages.
Reason : Restriction enzymes catalyse synthesis of
protective coat around bacterial cell that prevents the
attack of bacteriophage.
Both assertion and reason are true and reason
a)
is the correct explanation of the assertion

Both assertion and reason are true but reason

b) is not the correct explanation of the assertion

c) Assertion is true but reason is false

d) Both assertion and reason are false

 Discussion
● Restriction modification system provides defence against
foreign DNA such as those from bacteriophages.

Restriction modification
system

Restriction enzyme Modification enzyme

‘Restrict’ the multiplication of Add methyl groups to the

the viral/foreign DNA in the bacterial DNA, thus protecting
cells wherever present, by the bacterial DNA from getting
making several fragments of it. cleaved at the recognition sites
 5. Assertion: A bacterial cell with restriction enzymes
will be easily infected and lysed by bacteriophages.
Reason : Restriction enzymes catalyse synthesis of
protective coat around bacterial cell that prevents the
attack of bacteriophage.
Both assertion and reason are true and reason
a)
is the correct explanation of the assertion

Both assertion and reason are true but reason

b) is not the correct explanation of the assertion

c) Assertion is true but reason is false

d) Both assertion and reason are false

6. The rate of migration of DNA in an agarose gel is
affected by which of the following factor(s)?

I. Concentration of DNA in the sample

loaded on gel
II. Conformation of DNA molecule
III. Size of the DNA fragment
IV. Source from where DNA has been isolated

a) Only I b) II and III

c) I, II and IV d) II, III and IV

 Discussion

Gel electrophoresis - It is used for the separation of a

mixture of DNA fragments of varying sizes on agarose gel
matrix under the influence of electric field.

Cathode (-)
Sample Power supply
well

Gel
Anode (+)
 Discussion

● On application of electric field, the DNA molecules start

migrating towards the oppositely charged terminal (anode).
● The speed of migration is affected by the size and
conformation of the DNA molecules.
6. The rate of migration of DNA in an agarose gel is
affected by which of the following factor(s)?

I. Concentration of DNA in the sample

loaded on gel
II. Conformation of DNA molecule
III. Size of the DNA fragment
IV. Source from where DNA has been isolated

a) Only I b) II and III

c) I, II and IV d) II, III and IV

 7. Two DNA fragments are collected, i.e., A and B.
Assuming that A contains more base pairs than B,
select the incorrect statement with respect to gel
electrophoresis if the DNA samples A and B are
loaded in two wells.

a) A will stay near the cathode than B

B will migrate with a greater speed

b) compared to A towards the anode

A will migrate with a greater speed

c) compared to B towards the anode

Both A and B will form two distinct bands

d) corresponding to their molecular weights
 Discussion
DNA fragments

Gel electrophoresis

Molecules move through

Gel particles
the pores at a rate
inversely proportional to
Pores
their size

Visualization

DNA bands

Fig. Movement of DNA fragments

through the pores present in the gel
Discussion
 Discussion

Given that A has more base pairs than B.

● Since, they have different molecular
weights, they will form distinct bands
according to their molecular weights.
 7. Two DNA fragments are collected, i.e., A and B.
Assuming that A contains more base pairs than B,
select the incorrect statement with respect to gel
electrophoresis if the DNA samples A and B are
loaded in two wells.

a) A will stay near the cathode than B

B will migrate with a greater speed

b) compared to A towards the anode

A will migrate with a greater speed

c) compared to B towards the anode

Both A and B will form two distinct bands

d) corresponding to their molecular weights
 8. Plasmid pBR322 has PstI restriction enzyme site
within gene ampR that confers ampicillin resistance.
If this enzyme is used for inserting a gene for 𝛃-
galactoside production and the recombinant
plasmid is inserted in an E. coli strain:

It will be able to produce a novel protein with dual

a)
ability.

It will not be able to confer ampicillin resistance to

b) the host cell.

The transformed cells will have the ability to resist

c) ampicillin as well as produce 𝛃-galactoside

d) It will lead to lysis of host cell.

 Discussion

● Plasmid pBR322 has PstI restriction enzyme site

within gene ampR that confers ampicillin
resistance.
● If this enzyme is used for inserting a gene for 𝛃-
galactoside production and the recombinant
plasmid is inserted in an E. coli strain
● It will not be able to confer ampicillin resistance
to the host cell.
 Discussion

PstI 𝛃- galactoside

ampR ampR

pBR322 pBR322

Medium containing ampicillin Transformed bacterium

 8. Plasmid pBR322 has PstI restriction enzyme site
within gene ampR that confers ampicillin resistance.
If this enzyme is used for inserting a gene for 𝛃-
galactoside production and the recombinant
plasmid is inserted in an E. coli strain:

It will be able to produce a novel protein with dual

a)
ability.

It will not be able to confer ampicillin resistance to

b) the host cell.

The transformed cells will have the ability to resist

c) ampicillin as well as produce 𝛃-galactoside

d) It will lead to lysis of host cell.

 9. A person obtains transformants by inserting the desired gene
within the coding sequence of enzyme 𝛃-galactosidase. Which
of the following observations will help him separate out
recombinants from non-recombinants?

Non-recombinant colonies do not produce any colour

a) while recombinants give blue coloured colonies

Recombinant colonies do not produce any colour while

b) non-recombinants give blue coloured colonies

Both recombinants and non-recombinants produce

c) blue-coloured colonies

d) No colonies are formed due to insertional inactivation

 Discussion

GOI

LacZ
LacZ Ampr GOI
Ampr pUC8 pUC8

ori LacZ ori

β-galactosidase

White colonies
 Discussion

Non-recombinant Recombinant transformed

transformed bacteria bacteria

LacZ
LacZ GOI
Ampr
Ampr pUC8 pUC8

ori ori
 9. A person obtains transformants by inserting the desired gene
within the coding sequence of enzyme 𝛃-galactosidase. Which
of the following observations will help him separate out
recombinants from non-recombinants?

Non-recombinant colonies do not produce any colour

a) while recombinants give blue coloured colonies

Recombinant colonies do not produce any colour while

b) non-recombinants give blue coloured colonies

Both recombinants and non-recombinants produce

c) blue-coloured colonies

d) No colonies are formed due to insertional inactivation

10. Ti plasmid used in plant genetic engineering is a
plasmid found naturally in

a) Clostridium butyricum

b) Rhizobium leguminosarum

Agrobacterium tumefaciens
c)

d) Thermus aquaticus
 Discussion

● Tumor inducing (Ti) plasmid is present in Agrobacterium

tumefaciens which is a pathogen of several dicot plants.
● This bacteria naturally delivers its “T-DNA” piece of Ti
plasmid to the host.
○ This leads to transformation of normal plant cells
into tumor cells which produce chemicals required
by the bacterial pathogen.
 Discussion
This Ti plasmid has now been modified and engineered into a cloning vector which is no more
pathogenic to the plants but is still able to deliver genes of our interest into a variety of plants.

Plant
chromosomal
DNA
T-DNA Bacterial
Ti plasmid genome T-DNA
Crown
gall
Agrobacterium
tumefaciens Transformed
plant cell Plant
10. Ti plasmid used in plant genetic engineering is a
plasmid found naturally in

a) Clostridium butyricum

b) Rhizobium leguminosarum

c) Agrobacterium tumefaciens

d) Thermus aquaticus
11. ______A______ is a transformation method more
suitable for animals while ______B______ is more
suitable for plants.

a) A - Biolistics, B - microinjection

b) A - Gene gun, B - microinjection

c) A - Biolistics, B - gene gun

d) A - Microinjection, B - gene gun

 Discussion

The process by which exogenous DNA is transferred

into host cells is termed as transformation
 Discussion

Microinjection
● Recombinant DNA is directly injected into the nucleus of the cell.
● It is more suitable for animal cells.
 Discussion
Biolistics
● Cells are bombarded with high velocity micro-particles
of gold or tungsten coated with DNA.
● It is also known as a gene gun and is more suitable for
plant cells.
11. ______A______ is a transformation method more
suitable for animals while ______B______ is more
suitable for plants.

a) A - Biolistics, B - microinjection

b) A - Gene gun, B - microinjection

c) A - Biolistics, B - gene gun

d) A - Microinjection, B - gene gun

 12. Assertion: The bacterial cells must first be made
‘competent’ to take up foreign DNA.
Reason: DNA cannot pass through cell membranes
since it is a hydrophobic molecule.

a) Both the assertion and reason are true and the

reason is the correct explanation of assertion

Both the assertion and reason are true and reason

b) is not the correct explanation of assertion

c) Assertion is true but the reason is false

d) Both the assertion and reason are false

 Discussion

● Bacterial cells are made competent in order to

force them to take up foreign DNA.

● This is necessary because the cell membrane,

being selectively permeable, allows only
certain molecules to pass through it to
maintain a consistent internal environment.

● DNA cannot pass through cell membranes since

it is a hydrophilic molecule.
 Discussion
Arrangement of phospholipids in cell membrane

Water

Hydrophilic
(polar) head

Hydrophobic
(non polar) tails

Hydrophilic
(polar) head
Water
 Discussion
3.
2.

4.

1.

Steps to make bacterial

cells competent

5.
 12. Assertion: The bacterial cells must first be made
‘competent’ to take up foreign DNA.
Reason: DNA cannot pass through cell membranes
since it is a hydrophobic molecule.

a) Both the assertion and reason are true and the

reason is the correct explanation of assertion

Both the assertion and reason are true and reason

b) is not the correct explanation of assertion

c) Assertion is true but the reason is false

d) Both the assertion and reason are false

13. The stain that is used to view the bands of DNA
molecules on agarose gel is

a) bromophenol blue

b) ethidium bromide

c) safranin

d) methyl orange
 Discussion

● Agarose gel is the solidified gel which serves as the

matrix with definite pore size in gel electrophoresis.
 Discussion

Ethidium bromide is added in the agarose

gel once agarose powder is dissolved in
buffer and mixed thoroughly. The stain
gets dispersed throughout the gel.

Ethidium Bromide
(EtBr)
 Discussion

Agarose
gel

Ultraviolet
light source

Fig: Gel containing DNA bands after ethidium

bromide staining and UV exposure
13. The stain that is used to view the bands of DNA
molecules on agarose gel is

a) bromophenol blue

b) ethidium bromide

c) safranin

d) methyl orange
 14. Arrange the given steps involved in recombinant
DNA technology in the correct order.
A. Amplification of desired DNA sequence with PCR
modification
B. Ligation of gene of interest with the vector
C. Removal of RNA and proteins to purify DNA
D. Insertion of recombinant DNA into the host
E. Treatment of cell with cell wall digesting enzyme
followed by disruption of the cell membrane
F. Digestion of DNA of interest and vector with
suitable restriction enzyme

a) C→E→B→F→A→D b) E→C→A→F→B→D

c) E→C→A→B→F→D d) A→ E→C→B→D→F
 Discussion

Procedure of rDNA technology

Identifying gene of interest

Restriction enzymes cut at specific sites

Isolation of desired gene

Introduction of identified DNA into the host

Host with foreign DNA insert is selected

Transfer of desired DNA to the progeny

 Discussion

Procedure of rDNA technology

Gene of Interest (GOI)

Plasmid

Vector
Host DNA
 14. Arrange the given steps involved in recombinant
DNA technology in the correct order.
A. Amplification of desired DNA sequence with PCR
modification
B. Ligation of gene of interest with the vector
C. Removal of RNA and proteins to purify DNA
D. Insertion of recombinant DNA into the host
E. Treatment of cell with cell wall digesting enzyme
followed by disruption of the cell membrane
F. Digestion of DNA of interest and vector with
suitable restriction enzyme

a) C→E→B→F→A→D b) E→C→A→F→B→D

c) E→C→A→B→F→D d) A→ E→C→B→D→F
15. ________ is added to precipitate purified DNA.

a) Protease

b) Chilled ethanol

c) Lysozyme

d) Cellulase
 Discussion

● Ethanol will interfere with the DNA-water molecule

interactions.
● This will reduce the solubility of DNA in the water.

Chilled ethanol

DNA
precipitate
 Discussion

● As a result, DNA will precipitate out.

● A colder temperature will also help in faster

precipitation. Therefore, chilled ethanol
helps to precipitate the purified DNA.
15. ________ is added to precipitate purified DNA.

a) Protease

b) Chilled ethanol

c) Lysozyme

d) Cellulase
 16. Select the option that correctly identifies the given
statements as true or false.
I. The instrument used for PCR is called
thermocycler.
II. The copies of DNA synthesised by PCR
are not identical and each copy formed
has certain variations.
III. PCR amplifies DNA strands at an
exponential rate.

a) I - T, II - F, III - T b) I - T, II - T, III - F

c) I - F, II - T, III - F d) I - T, II - F, III - F
 Discussion
PCR or polymerase chain reaction is a technique used in molecular
biology to create several identical copies of a certain DNA segment.

1st cycle

2nd cycle

3rd cycle
 Discussion

● A thermocycler used to amplify small samples of DNA via

polymerase chain reaction.
● Thermocycler can easily switch the reaction conditions
between different temperatures at accurate time intervals.

Thermal cycler
 Discussion

● The formula to calculate the number of DNA

molecules formed after a given number of cycles is
2n (n = number of cycles of PCR).

● For example, if we were to do 32 rounds of PCR,

the number of molecules of double-stranded DNA
would be 232 . The value will reach up to billions.

Hence, it can be concluded that PCR amplifies

DNA at an exponential rate.
 16. Select the option that correctly identifies the given
statements as true or false.
I. The instrument used for PCR is called
thermocycler.
II. The copies of DNA synthesised by PCR
are not identical and each copy formed
has certain variations.
III. PCR amplifies DNA strands at an
exponential rate.

a) I - T, II - F, III - T b) I - T, II - T, III - F

c) I - F, II - T, III - F d) I - T, II - F, III - F
 17. If the “annealing” step is missed during PCR,
what would be its effect on the entire process?

I. DNA strands will not separate

II. Primers will not attach
III. DNA Polymerase would not work
IV. DNA will not extend

a) I & II b) II only

c) II, III & IV d) II & IV

 Discussion
PCR has three basic steps
DNA
DNA strands separated
Denaturation

96oC

Primer
Annealing
55-65 oC

dNTPs + DNA polymerase n cycles

Extension

72oC
 Discussion

● If the "annealing" step is missed, the

primers will not attach to the DNA
strands.
● DNA polymerase cannot function without a
primer.
● This is because DNA polymerase cannot
initiate polynucleotide chain synthesis.
 Discussion

● Taq polymerase enzyme adds the new

deoxyribonucleotide bases to the 3' end of the
primer.

● If primer is not present, then Taq polymerase

will have no place to add nucleotides and carry
out amplification. Therefore, DNA elongation
will not take place
 17. If the “annealing” step is missed during PCR,
what would be its effect on the entire process?

I. DNA strands will not separate

II. Primers will not attach
III. DNA Polymerase would not work
IV. DNA will not extend

a) I & II b) II only

c) II, III & IV d) II & IV

18. A gene of interest (GOI), amplified using PCR,
can be isolated using agarose gel electrophoresis by
____i____ and the DNA is extracted out. This
process is known as ____ii____.

i - cutting the portion of gel of the

a) desired band, ii - elution of DNA

i - scraping the gel of the desired band,

b) ii - extraction of DNA

i - extracting the DNA from the entire

c) gel, ii - digestion of DNA with
restriction enzymes

i - cutting the gel of the desired band,

d) ii - amplification of DNA
 Discussion

● A gene of interest (GOI), amplified using PCR, can be isolated

using agarose gel electrophoresis by cutting the portion of
gel of the desired band and the DNA is extracted out
 Discussion

● It is then treated with solvents and buffers and

filtered.

● This helps in the extraction of the purified DNA

fragment containing the gene of interest. This
process is known as the elution of DNA.
18. A gene of interest (GOI), amplified using PCR,
can be isolated using agarose gel electrophoresis by
____i____ and the DNA is extracted out. This
process is known as ____ii____.

i - cutting the portion of gel of the

a) desired band, ii - elution of DNA

i - scraping the gel of the desired band,

b) ii - extraction of DNA

i - extracting the DNA from the entire

c) gel, ii - digestion of DNA with
restriction enzymes

i - cutting the gel of the desired band,

d) ii - amplification of DNA
19. Sparged stirred-tank bioreactors have been
designed for

a) purification of product

addition of preservatives to the final

b) product

increased surface area for oxygen

c) transfer into culture

ensuring anaerobic condition in the

d) culture vessel
 Discussion

Increased surface
area for oxygen
transfer
Gas entrainment

Bubbles dramatically
increase the oxygen
transfer area

Sparged stirred-tank
19. Sparged stirred-tank bioreactors have been
designed for

a) purification of product

addition of preservatives to the final

b) product

increased surface area for oxygen

c) transfer into culture

ensuring anaerobic condition in the

d) culture vessel
20. The process of separation and purification of
expressed proteins from the cells grown on a large-
scale, before marketing is called

a) upstream processing

b) downstream processing

c) bioprocessing

d) biotechnology
 Discussion

Downstream processing

Centrifugation
Filtration
Sedimentation Chromatography

Separation Purification
Desired
product
 Discussion

● Biotechnology is the technology of using living organisms

or their parts for obtaining desired products, which can be
employed for commercial purposes.
● Bioprocessing, including both upstream and downstream
processing are components of biotechnology

Upstream Downstream Bioprocessing

Processing + Processing
20. The process of separation and purification of
expressed proteins from the cells grown on a large-
scale, before marketing is called

a) upstream processing

b) downstream processing

bioprocessing
c)

d) biotechnology