CBSE Term II Biology XII Biotechnology :
3
Principles and Processes
Multiple Choice Questions
1. (b) Restriction endonucleases cut both the strand of DNA
after recognising a specific nucleotide sequence for binding.
2. (c) DNA ligase joins or links two or more newly formed
DNA fragments.
3. (b) Vector or plasmid is a DNA molecule that can carry a
foreign DNA segment and can autonomously replicate.
Thus, it is used to transfer DNA into living cell.
4. (a) A–Eco RI, B–Bam HI, C–ori, D–amp R
5. (d) Correct sequence of step for the process of rDNA
technology is IV®III®I®II.
Assertion-Reasoning MCQs
1. (a) Both A and R are true and R is the correct explanation of A.
Maintenance of sterile (microbial contamination-free)
environment is essential in biotechnological processes to
enable growth of only the desired prokaryotic or eukaryotic
cells in large quantities for manufacture of biotechnological
products like antibiotics, vaccines, enzymes, etc.
2. (d) A is false, but R is true. A can be corrected as
Foreign DNA and vector DNA are cut with the help of same
restriction endonuclease. Ligase functions as glue to join the
fragments. It forms phosphodiester bonds between 3¢¾OH
end of one nucleotide and 5¢–phosphate end of another.
3. (a) Both A and R are true and R is the correct explanation of
A.
Fungal cell wall is made up of chitin. Thus, enzyme chitinase
is required for the digestion of fungal cell wall and isolation
of DNA from the cell.
Short Answer type Questions
1. A desired DNA segment can be introduced into a bacterial
cell in rDNA technology by the following methods.
(i) Microinjection
(ii) Biolistics/gene gun
(iii) Heat-shock method
(iv) Using disarmed pathogen vectors
2. There are certain features that are required to facilitate
cloning into a vector during RDT. These are
(i) Origin of replication (ori) is a specific DNA sequence
from where the process of replication begins. Thus, any
piece of DNA when linked to this sequence can be
made to replicate within the host cells. It also controls
the copy number of the linked DNA.
(ii) Selectable markers are genes which impart unique
characters to the vector. They help in identifying or
selecting the transformants and eliminating
non-transformants and hence, selectively permits only
the growth of the transformants, e.g. antibiotic
resistance.
3. Restriction enzymes are used for cutting of DNA at specific
locations during rDNA technology.
They are of two types
(i) Exonucleases which remove nucleotides from the ends
of DNA in one strand of the duplex.
1
(ii) Endonucleases which make cut at specific position
within DNA.
Each restriction endonclease functions by inspecting the
length of a DNA sequence. Once it finds specific recognition
sequence, it will bind to DNA and cut each of the two
strands of double helix at specific points in their
sugar-phosphate backbones.
Each restriction endonuclease recongnises a specific
palindromic nucleotide sequences in the DNA and cuts the
strand of DNA a little away from the centre of the
palindrome sites, but between the same two bases on the
opposite strands. This leaves single-stranded portion at the
ends termed as sticky ends.
4. There are certain features that are required to facilitate
cloning into a vector. These are
(i) Origin of Replication (ori) This is a sequence from
where replication starts and any piece of DNA, when
linked to this sequence can be made to replicate within
the host cells. This sequence is also responsible for
controlling the copy number of the linked DNA.
(ii) Selectable Marker It helps in identifying or selecting
transformants and eliminating non-transformants by
selectively permitting the growth of the transformants.
(iii) Cloning (recognition) Sites These are generally
required to link the foreign or alien DNA with the
vector.
Long Answer Type Questions
1. This amplification is done with the help of polymerase chain
reation.
Polymerase Chain Reaction (PCR) is defined as in vitro
process of DNA replication . This technique was developed
by Kary Mullis in 1985 . The basic requirements of a PCR
reaction are
(i) DNA template The double-stranded DNA that needs
to be amplified.
(ii) Primers These are chemically synthesised
oligonucleotides (short segment of DNA) that are
complementary to the regions of DNA template.
(iii) Enzymes Two commonly used enzymes in PCR
reaction are
l
Taq Polymerase It is isolated from a thermophilic
bacterium, i.e. Thermus aquaticus and has a property
to remain active during the high temperature-induced
denaturation of double-stranded DNA. It also helps in
the amplification of a segment of DNA .
l
Vent Polymerase It is isolated from Thermococcus
litoralis.
(iv) Nucleotide bases These are added by DNA polymerase
to the growing chain.
Three main steps involved in the PCR technique are
Step I Denaturation The double-stranded DNA is denatured
by using high temperature of 95° C for 15 seconds.
Now each separated single strand acts as a template for
DNA synthesis.
2 CBSE Term II Biology XII

Step II Annealing Two sets of oligonucleotide primers are annealed (hybridised) to the separated single-strands. This step is carried
out at a slightly lower temperature (40-60° C).
Step III Extension The thermostable enzyme Taq DNA polymerase used in this reaction, extends the primers by adding dNTPs
(deoxynucleoside triphosphates) complementary to those of the template DNA. Mg 2 + is required as a cofactor for thermostable
DNA polymerase.
These steps are repeated many times in order to obtain several copies of desired DNA.
Region to be amplified

5¢ 3¢
3¢ 5¢ dsDNA
Heat Denaturation

5¢ 3¢
3¢ 5¢
Primers
5¢ 3¢ Annealing
3¢ 5¢ of primers

DNA polymerase
(Taq polymerase)
+ deoxynucleotides
5¢ 3¢
3¢ 5¢
Extension
5¢ 3¢ of primers
3¢ 5¢

30 cycles

Amplified
(~1 billion times)

Polymerase Chain Reaction (PCR)

2. Bioreactors 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.

Increased surface
Motor area for
Acid/Base
for pH control oxygen transfer Gas entrainment
Foam braker

Steam for Flat bladed impeller

sterilisation
Culture broth
Bubbles/dramatically
increase the oxygen
transfer area

Sterile air
(a) (b)

(a) Simple stirred-tank bioreactor, (b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged
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.
The components of a bioreactor are
(i) An agitator system (ii) An oxygen delivery system
(iii) A foam control system (iv) A temperature control system
(v) pH control system (vi) A sampling port to withdraw culture periodically.
The most commonly used bioreactors are of stirring type. Stirring type bioreactors are further of two types, i.e. simple and
sparged. A simple stirred-tank bioreactor is usually cylindrical or with a curved base to facilitate even mixing of reactor contents.
The sparged-stirred-tank bioreactor also facilitates the mixing of components and ensures oxygen availability throughout the
bioreactor. Alternatively, air is bubbled through the reactor. Which sterile air bubbles are sparged.