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710

BIOLOGY

Chapter

33

Biotechnology:
Principles and
Processes

Biotechnology refers to the technology using biology, which has
application in agriculture, food processing industry, bioremediation,
medicine diagnostic, waste treatment and energy production
...

The European Federation of Biotechnology (EFB) defines
Biotechnology as ‘the integration of natural science and organisms,
cells, parts thereof, and molecular analogues for products and
services’
...

² In vitro fertilisation (‘test-tube’ baby programme)
² Synthesis and using of a gene
² Preparation of a DNA vaccine
² Correcting a defective gene

introducing undesirable genes into target organism
...
Therefore, for the multiplication of any alien
piece of DNA in an organism, it needs to be a part of a
chromosome which has specific sequence called ‘origin of
replication’
...


The two core techniques that enabled birth of modern biotechnology
are:
(a) Genetic engineering: It is the technique in which the
genetic material (DNA and RNA) is chemically altered and
introduced into host organisms to change their phenotype
...

(b) Maintenance of microbial contamination free surrounding
(sterile ambience): It is necessary in chemical engineering
processes to enable growth of only desired microbe/
eukaryotic cell in large quantities
...


Development of the Principle of Genetic Engineering
y Traditional hybridization procedures used in plant and animal
breeding lead to inclusion of undesirable genes along with
desired genes
...
This is known as cloning
...

y The cutting of DNA at specific locations became possible
with the discovery of the so-called “Molecular scissors”
i
...
, restriction enzymes
...

y Stanley Cohen and Herbert Boyer in 1972 isolated the
antibiotic resistance gene by cutting out a piece of DNA
from a plasmid
...
These
recombinant DNA molecules are then introduced into host
cells, where they can be propagated and multiplied
...

² Maintenance of introduced DNA in the host and transfer
of the DNA to its progeny
...
Restriction Enzymes (‘Molecular Scissors’)
y Restriction enzymes are molecular scissors used in molecular
biology for cutting DNA sequences from a specific site
...

y The enzyme recognises a specific six box pair sequence
known as the recognition sequence and cut the sequence at
a specific site
...
It always cuts
DNA molecules at a particular point by recognizing a specific
sequence of six base pairs
...

y Today more than 900 restriction enzymes have been isolated
from over 230 strains of bacteria
...
These DNA fragments will hydrogen bond to
each other due to their complementary sequences
...

5' - GAATIC - 3'

Eco RI

5' - G

3' - CTTAAG - 5'

Digest

3' - CTTAA

AATTC - 3'
5'

G - 5'

sticky-ends

5' - CCCGG - 3'

Smal

5' - CCC

GGG - 3

3' - GGGCC

Digest

3' - GGG

CCC - 5'

- 5'

blunt ends

y Restriction enzymes belong to a class of enzymes called
nucleases
...

(ii) Endonucleases: It is a type of restriction enzymes
that make a cut within the DNA at a specific site
...

It is commonly used to make a cut in the sequence to
obtain DNA fragments with sticky ends
...
These ends are later on joined by enzyme
ligase
...


Fig
...

y Palindromes: The palindrome in DNA is a sequence of
base pairs that reads the same on the two strands in 5' → 3'
direction and in 3' → 5' direction
...
g
...
This leaves single stranded
overhanging stretches at the ends
...
This stickiness facilitates action of the enzyme DNA
ligase
...

Convention of naming restriction enzymes
y The first letter of the name comes from the genus
...


BIOLOGY

yy The Roman letter followed is the order of discovery
...
g
...
It
is a duplicate or look-alike carrying the same genetic
signature
...

yy Molecular cloning is carried out in-vitro where a specific
fragment of DNA is isolated from an organism ‘donor’ and
introduced into a ‘plasmid’ that replicates in a ‘host’ cell
making multiple copies of that DNA fragment
...

yy The most commonly used vectors are:
(i) Plasmids (5-10 kb): Plasmid is an extra-chromosomal
DNA molecule in bacteria that is capable of replicating,
independent of chromosomal DNA
...
They are mostly found inside
certain bacterial species such as E
...
Some plasmids
have only 1-2 copies per cell
...
E
...
pUC18 (2686 bp, in host it replicates
500 copies per cell and contains several RE sites)
...
Cloning Vectors (Vehicles for Cloning)

Remarks

EcoRI

E
...

yy These DNA fragments can be separated by a technique called
gel electrophoresis
...
They can be
separated by forcing them to move towards the anode
under an electric field through a medium/matrix
...
Agarose is a
natural polymer extracted from sea weeds
...

Hence, the smaller sized fragment move farther
...

²² Separated bands of DNA are cut out from agarose gel
...

²² These DNA fragments are used in recombinant DNA
by joining them with cloning vectors
...
 

Fig
...
Hence, it can be used to
transfect and create several copies of the DNA fragment
of interest by replicating several times in bacteria
...

E
...
Lambda (able to replace 1/3 of the chromosome
with large pieces of foreign DNA), M13a
Note: Alien DNA linked with the vector multiply its number
equal to the copy number of the plasmid or bacteriophage
...


Sal I

yy Ori is a DNA sequence in a genome from where replication
initiates
...


orl

yy Any piece of DNA when linked to ori can be made to replicate

rop

within the host cells
...

yy So, for getting many copies of the target DNA it should be

Pvu II

cloned in a vector whose origin support high copy number
...
  E
...

yy The process through which a piece of DNA is introduced in
a host bacterium is called transformation
...
are
considered useful selectable marker for E
...
The normal
E
...

yy Thus vector should incorporate a selectable marker (antibiotic
resistancegene), which will allow to select those host cells
that contain the vector from amongst those that do not
...

yy The vector must have very few (preferably single) recognition
sites
...

yy Therefore, the foreign DNA is ligated at a restriction site
present in one of the two antibiotic resistance genes
...
coli is pBR 322
...




Identification of recombinants:
yy Insertional inactivation
The most efficient method of screening for the presence
of recombinant plasmids is based on the principle that the
cloned DNA fragment disrupts the coding sequence of a
gene
...

yy If a foreign DNA ligated or inserted at the Bam H I site
of tetracycline resistance gene in the vector pBR322, the
recombinant plasmid will lose tetracycline resistance
...

yy The recombinant plasmids lose tetracycline resistance due
to insertion of foreign DNA
...

yy Then these transformants are transferred on tetracycline
medium
...
But, non-recombinants will grow
on the medium containing both the antibiotics
...

yy Selection of recombinants due to inactivation of antibiotics
requires simultaneous plating on two plates having different
antibiotics
...




BIOLOGY
yy Similarly, Retroviruses have also been disarmed and are now
used to deliver desirable genes into animal cells
...


Alternative selectable marker
yy A recombinant DNA is inserted within the coding sequence
of an enzyme, β-galactosidase
...
This results into
inactivation of enzyme, which is referred to as insertional
inactivation
...

yy Presence of insert results into insertional inactivation of the
β-galactosidase and the colonies do not produce any colour,
these are referred to as recombinant colonies
...

Agrobacterium tumifaciens is a pathogen of many dicot
plants
...
Competent Host (Introduction of DNA into Host Cells)
yy Competent host is essential for transformation with
recombinant DNA
...

yy Cells that are able to take up DNA are called competent
cells
...

Calcium increases the efficiency with which DNA enters
the bacterium through pores in its cell wall
...
This enables the
bacteria to take up the recombinant DNA
...

²² Biolistics (gene gun): In this, cells are bombarded with
high velocity micro-particles of gold or tungsten coated
with DNA
...

²² ‘Disarmed pathogen’ vectors (Agrobacterium
tumifaciens) which when infects the cell transfer the
recombinant DNA into the host
...


yy In this, specific nucleotide sequences are cut’ from the DNA
of an organism and “pasted” into plasmids
...

yy It is then inserted into bacteria
...

yy It includes the following steps:
1
...

2
...

3
...

4
...

5
...

6
...

7
...


EBD_7051

714

715

Biotechnology: Principles and Processes

1
...

y Therefore, to get pure DNA (free from other macromolecules), the bacterial cells/plant or animal tissue are
treated with enzymes such as lysozyme (for bacterial cell),
cellulase (for plant cells), chitinase (for fungal cell) etc
...

y Genes (DNA) are interwined with proteins such as histones
...

² Proteins are removed by treatment with protease
...


y The enzyme utilises building blocks dNTPs (deoxynucleotides)
to extend the primer
...


E
...
Fragmentation of DNA by Restriction Enzymes
y DNA is cut into fragments with the help of restriction
enzymes
...

y Agarose gel electrophoresis is employed to check the
progression of a restriction enzyme digestion
...
The process
is repeated with the vector DNA also
...

y This creates recombinant DNA
...


3
...
Several identical copies of it can be synthesized
in vitro
...

y It is extensively used in the process of gene manipulation
...

y Primers are small chemically synthesized oligonucleotides
that are complementary to the regions of DNA
...

² Denaturation: The double stranded DNA molecules
are heated to a high temperature (94˚C) so that the two
strands separate into a single stranded DNA molecule
...
Each strand acts as
template for DNA synthesis
...
This
step is carried out at a lower temperature depending on
the length and sequence of the primers
...

² Extension of primers: DNA polymerase (Taq
polymerase) extends the primers using the nucleotides
provided in the reaction
...


BIOLOGY

y This process is repeated over several cycles to obtain multiple
copies of rDNA fragment
...
This result recombinant DNA (rDNA)
...

y The cells with foreign genes may be grown on a small scale
in the laboratory
...

y The cells can also be multiplied in a continuous culture
system
...
It maintains the
cells more physiologically active and so produces a larger
biomass leading to higher yields of desired protein
...
Here,
the raw materials are biologically converted into specific
products, enzymes etc
...

y They provide optimal conditions to obtain the desired product
by providing the optimum temperature, pH, vitamin, oxygen
etc
...


Fig
...
The method is called reverse transcriptase
PCR (RT-PCR)
...

² It is mixed with reverse transcriptase and a primer that
will anneal to the 3’end of the RNA of interest
...


4
...
The
recipient cell makes itself competent to receive and take up
DNA present in the surrounding
...
Recipient cells take up DNA present in
its surrounding
...
coli cells,the
host cells become ampicillin-resistant cells
...


5
...


Fig
...
The stirrer facilitates
even mixing and oxygen availability
...
The bioreactor has
♦ An agitator system
♦ An oxygen delivery system
♦ A foam control system
♦ A temperature control system
♦ pH control system
♦ Sampling ports (for periodic withdrawal of the
culture)
...

On the other hand, stirred tank bioreactors are used for
large scale production of biotechnology products
...

(i) Small volumes of culture can be taken out from the
reactor for sampling or testing
...

(iii) It has a control system that regulates the temperature
and pH
...
Downstream Processing
y The products so obtained undergo a series of processes
before putting them in market as a finished product
...
This is known
as down streaming processing
...

y The product is subjected to various processes in order to
separate and purify the product
...

y The downstream processing and quality control testing vary
from product to product
...
e;
restriction enzymes cloning
vectors and host organisms

Restriction Enxymes

By following this only
desired microbes/
eukaryotic cell can
grow in cultures

Maintenance of sterile
ambience

It include to core techniques
which is used in modern
biotechnology

Principles of
Biotechnology

Deals with techniques of using live organisms
to get desired products of human welfare

Biotechnology : Principles and Processes

Obtaining the foreign
Gene product

Amplification of Gene of
Interest using PCR

Cutting of DNA at
specific locations

Isolation of the Genetic
Material (DNA)

Process of Recombinant
DNA technology

718

EBD_7051

BIOLOGY

Biotechnology : Principles and Processes

1
...


3
...


5
...


7
...


9
...

(b) hybrid produced by sexual means
...

(d) heterozygous plant produced by sexual means
...

(b) monoclonal antibody production
...

(d) All of the above
Which structure involved in genetic engineering?
(a) Plastid
(b) Plasmid
(c) Codon
(d) None of these
Genetic engineering aims at
(a) destroying wild gene
(b) preserving defective gene
(c) curing human disease by introducing new gene
(Haemophilia)
(d) all the above
Ti-plasmid used in genetic engineering has been modified by
(a) adding tumour forming genes
...

(c) adding genes for endonucleases
...

Which of the following technique is used for the separation
of DNA fragments ?
(a) Gel electrophoresis
(b) Chromatography
(c) Transformation
(d) Transduction
Which of the following is a tool of recombinant DNA
technology ?
(a) Cloning vectors
(b) Ligase enzymes
(c) Restriction enzymes
(d) All of the above
Which of these is not correctly matched ?
(a) Gene gun—biolistic gun
(b) Plasmids—extrachromosomal DNA
(c) DNA ligase—Biological scissors
(d) Bacteriophages—viruses
...
A single strand of nucleic acid tagged with a radioactive
molecule is called :
(a) Vector
(b) Selectablemarker
(c) Plasmid
(d) Probe
11
...
Polyethylene glycol method is used for
(a) biodiesel production
(b) seedless fruit production
(c) energy production from sewage
13
...


15
...


17
...


19
...

(b) include genes for bioluminescence
...

(d) Both a and b
Rennin used in cheese industry is
(a) antibiotic
(b) alkaloid
(c) enzyme
(d) inhibitor
Restriction endonucleases
(a) are enzymes that process pre-RNA’s
...

(c) protect bacterial cells from viral infection
...

(b) every organism has the same amount of DNA
...

(d) DNA has a consistent structure in all organisms
...


21
...


23
...


25
...


27
...

(b) the function of the gene is known
...

(d) the gene has been isolated and copied
...
The next step is to use
(a) restriction enzymes that break the DNA into small pieces
at known points
...

(c) ligases that make the DNA stick together
...

An enzyme that joins the ends of two strands of nucleic acid
is a
(a) polymerase
(b) synthetase
(c) helicase
(d) ligase
Introduction of one or more genes into an organism which
normally does not possess them or their deletion by using
artificial means (not by breeding) comes under
(a) molecular biology
(b) cytogenetics
(c) genetic hybridization
(d) genetic engineering
Which one of the following pairs is not correctly matched
(a) Plasmid – Small piece of extrachromosomal DNA in
bacteria
(b) Interferon – An enzyme that interferes with DNA
replication
(c) Cosmid – A vector for carrying large DNA fragments into
host cells
(d) Myeloma – Antibody - producing tumour cells
DNA fragments are separated using gel electrophoresis
(a) because DNA is pulled through the gel toward the
negative end of the field
...

(c) to identify and isolate DNA fragments
...


28
...


30
...


32
...


34
...


36
...

(b) hybridization between DNA and transcription factors
...

(d) synthesizing cDNA molecules from mRNA templates
...

(b) centromeres
...

(d) an origin of replication
...

(b) a gene from one organism has been inserted into a vector
and successfully introduced into a host cell
...

(d) All of the above
Recombinant DNA can be transferred into host cell by
(a) growing the host cell in growth medium containing
ampicillin
...

(c) treating cells with calcium ions or electrical pulses to
increase cell permeability
...

A genetic marker is a
(a) place where a restriction enzyme cuts DNA
...

(c) nucleotide sequence near a particular gene
...


EBD_7051

BIOLOGY

720

Biotechnology : Principles and Processes
37
...


39
...


41
...


43
...


Expression vectors are different from other vectors because
they
(a) contain drug resistance markers
...

(c) contain regulatory regions that permit the cloned DNA
to produce a gene product
...

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
A genomic library is
(a) where you look to find out how to make recombinant
DNA
...

(c) all the genes contained in one kind of cell
...

Electrophoresis is used to
(a) separate fragments of DNA
...

(c) cut DNA into fragments
...

Which of the following is not necessary to execute a
polymerase chain reaction successfully?
(a) All four DNA bases
(b) Short DNA base primers
(c) DNA polymerase
(d) DNA library
Which of the following statements about restriction enzymes
is false?
(a) They work on DNA extracted from all types of organisms
...

(c) They come in many varieties, each with its own DNA
target sequence
...

DNA ligases are enzymes that can be used to
(a) chop a large DNA molecule into small fragments
...

(c) insert the DNA from one species into the DNA of another
species
...

Imagine a gel through which DNA fragments have moved in
response to an applied electrical current
...

(b) longest fragments of DNA
...

(d) ligase used to bind the DNA fragments together
...
First restriction endonuclease enzyme was discovered in
(a) PPLO
(b) E
...
A biologist intends to use a polymerase chain reaction to
perform a genetic task
...

(b) clone a gene
...

(d) isolate DNA from a living cell
...
In genetic engineering, genes can be inserted from one
organism into another or back into the original organism using
which of the following techniques?
(a) Polymerase chain reaction
(b) Gene gun
(c) DNA hybridization
(d) Gel electrophoresis
48
...
Identify the correct match for the given apparatus
...
Recombinant DNA molecules were first of all synthesized by
Paul Bery using DNA of
(a) TMV and Salmonella
(b) Virus SB-40 and E
...


2
...


4
...


6
...


PCR and Restriction Fragment Length Polymorphism are the
methods for :
(a) Study of enzymes
(b) Genetic transformation
(c) DNA sequencing
(d) Genetic Fingerprinting
Which one is a true statement regarding DNA polymerase
used in PCR
(a) It is used to ligate introduced DNA in recipient cell
(b) It serves as a selectable marker
(c) It is isolated from a virus
(d) It remains active at high temperature
Biolistics (gene-gun) is suitable for
(a) DNA finger printing
...

(c) Transformation of plant cells
...

In genetic engineering, the antibiotics are used
(a) as selectable markers
...

(c) to keep the cultures free of infection
...

Which of the following forms the basis of DNA finger printing?
(a) The relative proportions of purines and pyrimidines in
DNA
...

(c) The relative difference in the DNA occurrence in blood,
skin and saliva
...

The first clinical gene therapy was given for treating
(a) chicken pox
(b) diabetes mellitus
(c) rheumatoid arthritis
(d) adenosine deaminase deficiency
Which one of the following represents a palindromic sequence
in DNA?
(a) 5' - GAATTC - 3'
3' - CTTAAG - 5'
(b) 5' - CCAATG - 3'
3' - GAATCC - 5'
(c) 5' - CATTAG - 3'
3' - GATAAC - 5'
(d) 5' - GATACC - 3'
3' - CCTAAG - 5'

8
...


10
...


12
...


14
...
CGTTCG
...
ATGGTA
...
GATATG
...
CTACTA
...
GAATTC
...
CTTAAG
...
CACGTA
...
CTCAGT
...
What does
co
...


723

Choose the correct option
...


Which one of the following option is correct for A, B, C
and D ?
Foreign DNA

Vector
DNA
(plasmid)

A

B
join foreign
DNA to plasmid

C

D
E
...


(a) A-Exonuclease; B-Endonuclease; C-DNA ligase;
D-Transformation
(b) A-Exonuclease; B-Exonuclease; C-DNA ligase;
D-Transformation
(c) A-Exonuclease; B-En donuclease;C- Hydrolase;
D-Transduction
(d) A-Restriction endonuclease; B-Restriction endonuclease;
C-DNA ligase; D-Transformation
Identify the correct match for the given apparatus
...
Find out the pairs, which are correctly matched
...
PCR
1
...
Bioreactor
2
...
Gene gun
3
...
Eco RI
4
...

(a) A ® (4); B ® (1); C ® (2); D ® (3)
(b) A ® (2); B ® (1); C ® (4); D ® (3)
(c) A ® (4); B ® (1); C ® (3); D ® (2)
(d) A ® (1); B ® (4); C ® (2); D ® (3)
19
...

Column I
Column II
A
...
Agrobacterium
tumifaciens
B
...
Cancerous cells
C
...
Recombinant DNA
D
...
Restriction enzyme
(a) A ® (4); B ® (1); C ® (2); D ® (3)
(b) A ® (2); B ® (1); C ® (4); D ® (3)
(c) A ® (4); B ® (1); C ® (3); D ® (2)
(d) A ® (1); B ® (4); C ® (2); D ® (3)
20
...

Column I
Column II
A
...
Running of DNA
fragments on gel
B
...
Cutting of DNA
into fragments
C
...
Transfer of DNA
fragment from gel to
nitrocellulose disc
D
...
Searching for desired
DNA fragments
(a) A ® (4); B ® (1); C ® (2); D ® (3)
(b) A ® (2); B ® (1); C ® (4); D ® (3)
(c) A ® (4); B ® (1); C ® (3); D ® (2)
(d) A ® (1); B ® (4); C ® (2); D ® (3)

21
...


23
...

(a) (1) and (2)
(b) (2) and (3)
(c) (1) and (4)
(d) (1), (2), (3) and (4)

DIRECTIONS for Qs
...
Each
question has 4 choices (a), (b), (c) and (d) out of which ONLY ONE
is correct
...


24
...

Statement 2 : Proteins which can be synthesized only by
genetic engineering are effective against viruses
...


Statement 2 : Both bacteria and yeast multiply very fast to
form huge population which express the desired gene
...


2
...


4
...


Rising of dough is due to
(a) multiplication of yeast
(b) production of CO2
(c) emulsification
(d) hydrolysis of wheat flour starch into sugars
An enzyme catalysing the removal of nucleotides from the
ends of DNA is
(a) endonulease
(b) exonulease
(c) DNA ligase
(d) Hind II
The transfer of genetic material from one bacterium to another
through the mediation of a vector like virus is termed as
(a) transduction
(b) conjugation
(c) transformation
(d) translation
Which of the given statement is correct in the context of
observing DNA separated by agarose gel electrophoresis?
(a) DNA can be seen in visible light
...

(c) Ethidium bromide stained DNA can be seen in visible
light
...

'Restriction' in restriction enzyme refers to
(a) cleaving of phosphodiester bond in DNA by the enzyme

Statement 1: In recombinant DNA technology, human genes
are often transferred into bacteria (prokaryotes) or yeast
(eukaryote)
...


7
...


9
...
coli
In agarose gel electrophoresis, DNA molecules are separated
on the basis of their
(a) charge only
(b) size only
(c) charge to size ratio
(d) All of these
The most important feature in a plasmid to be used as a vector
is
(a) Origin of replication (Ori)
(b) presence of a selectable marker
(c) presence of sites for restriction endounclease
(d) its size
While isolating DNA from bacteria, which of the following
enzymes is not used?
(a) Lysozyme
(b) Ribonuclease
(c) Deoxyribonuclease
(d) Protease

EBD_7051

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724

Biotechnology : Principles and Processes
10
...


12
...


14
...


16
...


Which of the following has popularised the PCR (Polymerase
Chain Reaction)?
(a) Easy availability of DNA template
(b) Availability of synthetic primers
(c) Availability of cheap deoxyribonucleotides
(d) Availability of 'Thermostable' DNA polymerase
An antibiotic resistance gene in a vector usually helps in the
selection of
(a) competent cells
(b) transformed cells
(c) recombinant cells
(d) None of these
Significance of heat shock method in bacterial transformation
is to facilitate
...
However, the transformed
cells did not produce the desired protein
...
who among the following was awarded the Noble Prize for the
development of PCR technique?
(a) Herbert Boyer
(b) Hargovind Khurana
(c) Kary Mullis
(d) Arthur Kornberg
19
...
DNA fragments generated by the restriction endonucleases
in a chemical reaction can be separated by :
[2013]
(a) Polymerase chain reaction
(b) Electrophoresis
(c) Restriction mapping
(d) Centrifugation
21
...
Genes of interest can be selected from a genomic library by
using
[NEET Kar
...
During the process of isolation of DNA, chilled ethanol is
added to
[NEET Kar
...
An analysis of chromosomal DNA using the Southern
hybridization technique does not use:[2014]
(a) Electrophoresis
(b) Blotting
(c) Autoradiography
(d) PCR
25
...


27
...


29
...


Which vector can clone only a small fragment of DNA? [2014]
(a) Bacterial artificial chromosome
(b) Yeast artificial chromosome
(c) Plasmid
(d) Cosmid
Commonly used vectors for human genome sequencing are:
[2014]
(a) T-DNA
(b) BAC and YAC
(c) Expression Vectors
(d) kT/A Cloning Vectors
The cutting of DNA at specific locations became possible
with the discovery of
[2015 RS]
(a) Probes
(b) Selectable markers
(c) Ligases
(d) Restriction enzymes
The DNA molecule to which the gene of interest is integrated
for cloning is called
[2015 RS]
(a) Vector
(b) Template
(c) Carrier
(d) Transformer
Which of the following is not a feature of the plasmids? [2016]
(a) Independent replication
(b) Circular structure
(c) Transferable
(d) Single - stranded

31
...


33
...


35
...


The taq polymerase enzyme is obtained from
[2016]
(a) Thermus aquaticus
(b) Thiobacillus ferroxidans
(c) Bacillus subtilis
(d) Pseudomonas putida
Which of the following is a restriction endonuclease?[2016]
(a) Hind II
(b) Protease
(c) DNase I
(d) RNase
The DNA fragments separated on an agarose gel can be
visualised after staining with :
[2017]
(a) Acetocarmine
(b) Aniline blue
(c) Ethidium bromide
(d) Bromophenol blue
The process of separation and purification of expressed
protein before marketing is called:
[2017]
(a) Downstream processing
(b) Bioprocessing
(c) Postproduction processing
(d) Upstream processing
A gene whose expression helps to identify transformed cell is
known as :
[2017]
(a) Vector
(b) Plasmid
(c) Structural gene
(d) Selectable marker
What is the criterion for DNA fragments movement on agarose
gel during gel electrophoresis?
[2017]
(a) The smaller the fragment size, the farther it moves
(b) Positively charged fragments move to farther end
(c) Negatively charged fragments do not move
(d) The larger the fragment size, the farther it moves

EBD_7051

BIOLOGY

726

Biotechnology : Principles and Processes

727

Hints & Solutions
EXERCISE - 1
1
...

10
...


12
...

19
...


21
...

23
...

25
...


(a) 2
...

(b) 4
...

(b) 6
...

(c) 9
...

(d) For gene transfer into the host cell without using vector
microparticles made of tungsten and Gold coated with
foregin DNA are bombarded into target cells at a very
high velocity
...
DNA into
plant cells but the presence of rigid plant cell wall acts as
a barrier to uptake
...
Polyethylene glycol
mediated DNA uptake is a direct gene transfer method
that utilizes the interaction between polyethylene glycol,
naked DNA, salts and the protoplast membrane to effect
transport of the DNA into the cytoplasm
...
(c) 15
...
(d) 17
...
(c)
(d) The fact that DNA is structured the same way in all known
organisms means that similar methods can be used to
study the hereditary material
...
The probes bond (hybridize) with the target
sequence wherever it occurs in the DNA sample
...

(d)
(a) Once isolated, DNA can be cut into fragments using
restriction enzymes
...

(d)
(d) Genetic engineering is the science of gene manipulation
...
The aim is to introduce new
characteristics or attributes physiologically or physically,
such as making a crop resistant to a herbicide, introducing
a novel trait, or producing a new protein or enzyme, along
with altering the organism to produce more of certain
traits
...
coli
...


28
...

31
...

33
...

35
...

37
...


39
...

41
...

43
...


45
...


(c) DNA fragments migrate toward the positive end of the
electrical field with the smallest fragments migrating the
fastest
...
(d)
(d) A prokaryotic vector needs an origin of replication to be
propagated in a prokaryotic cell
...
The Bt cotton variety contains a foreign gene
obtained from Bacillus thuringiensis
...

(d)
(d) All of these molecules can serve as vectors for cloned
DNA
...
Coating DNA with lipids (not
carbohydrates) is used to introduce recombinant DNA
molecules into host cells, injecting DNA (not protein) is
a method used to place recombinant DNA into best cells
...

Expression vectors are unique because they contain
regulatory sequences that allow the cloned gene to be
expressed in the host cell
...
It has been
designed for availability of oxygen throughout the
process
...

(a) Electrophoresis separates DNA fragments by size
...

(c) Ligases are used to connect short DNA fragments to
form longer segments, a process essential for inserting
DNA segments into vectors
...

(c)
(b) PCR is a way to dramatically increase the numbers of a
particular sequence of DNA (or an entire gene)
...


47
...


49
...

(b) Besides the gene gun, other methods of genetic transfer
between cells or organisms include the use of vectors
such as viruses and plasmids
...
(b)

EXERCISE - 2
1
...

3
...


5
...


7
...


9
...


(d)
(d) Taq polymerase are extracted from a thermophilic bacterial
and so reman active at high temperature
...

(a) Antibiotics are powerful medicines that fight bacterial
infections
...
In genetic engineering, the antibiotics are
used as selectable markers
...
The first clinical gene therapy
was given for treating adenosine deaminase deficiency
...

Adenosine deaminase deficiency, also called ADA
deficiency or ADA-SCID is an autosomal recessive
metabolic disorder that causes immunodeficiency
...

(a) A palindromic sequence is a nucleic acid sequence (DNA
or RNA) that is the same whether read 5' (five-prime) to 3'
(three prime) on one strand or 5' to 3' on the
complementary strand with which it forms a double helix
...

(d) Plants developed by genetic engineering are called
transgenic plants or genetically modified crops from which
genetically modified food is produced
...

So, by consuming them there is a danger of entry of
viruses and toxins causing different types of allergies
and other health hazards to human beings
...
DNA ligase is
an enzyme that is able to join together two portions of
DNA and therefore plays an important role in DNA repair
...

(c) Palindromic sequences in DNA molecule are group of
bases that forms the same sequence when read in both
forward and backward direction
...


11
...


13
...

15
...

24
...


(a) Restriction endonucleases are enzymes that makes cuts
at specific positions within the DNA molecule
...
They recognise specific base
sequence at palindrome sites in DNA duplex and cut its
strands
...
coli and a part of restriction modified system
...

(d) DNA finger printing technique was first developed by
Alec Jeffreys, Wilson and Thien in 1984
...

(a) In industries, fermentation is carried out in special devices
(fermentation tank) called bioreactors
...
(d) 17
...
(a) 19
...
(b)
(d) 22
...
(c)
(c) Interferons are proteins that are effective against most
viruses
...
The proteins interact with adjacent cells and make
them resistant to virus attack
...

Interferons control the multiplication of virus particles
by inhibiting their protein synthesis
...


EXERCISE - 3
Exemplar Questions
1
...


3
...
It is used to make foods like idli,
dosa, bread, etc
...

(ii) Endonucleases cuts at specific position within the
DNA
...

(a) The process by which genetic material (DNA) is
transferred from one bacterium to another through the
mediation of a vector like virus is known as transduction
...


EBD_7051

BIOLOGY

728

Biotechnology : Principles and Processes

4
...


(b)

6
...


(b)

8
...


(c)

10
...

Translation is the process in which cellular ribosomes
create proteins
...

By the process of gel electrophoresis, DNA fragments
are separated and are visualised after staining the DNA
with ethidium bromide followed by exposure to UVradiation
...

The restriction enzymes are known as 'molecular scissors
and are responsible for cutting DNA
...
They are
of two types:
(i) Exonucleases : Cut DNA at the ends
(ii) Endonucleases : Make cuts at specific positions
within the DNA
...

Restriction enzymes and DNA ligases are used to make a
stable recombinant DNA molecule, with DNA fragments
that has been spliced together from two different
organisms
...
It means, the
smaller the fragment size, the faster it will move
...

This is because
(i) Ori is a DNA sequence that is responsible for
initiation of replication
...

(ii) It also controls the copy numbers of the linked
DNA
...
Since, the DNA is enclosed
within the membranes, we need to break the cell to release
DNA along with other macromolecules such as RNA,
proteins, polysaccharides and also lipids
...

The amplification of specific DNA sequences is carried
out in vitro called the Polymerase Chain Reaction (PCR)
...


729
11
...


13
...


15
...


17
...
Selectable markers help in identifying
and eliminating non-transformants and selectively
permits the growth of the transformants
...
g
...

(c) In the process of chemical method, the cell is treated with
specific concentration of a divalent cation such as
calcium to increase pore size in cell wall
...
this process is called heat shock method
...

(a) DNA ligase or sealing enzymes are also called genetic
gum
...
Thus they help in joining gaps in DNA
fragments
...

(c) Agrobacterium tumefaciens acts as pathogen for several
dicot plants
...

(c) In polymerase chain reaction polymerisation or extension
step is catalysed by Taq polymerase enzyme
...
Each separated single stranded
strand now acts as template for DNA synthesis
...
Primers
act as initiators of replication
...

A thermostable DNA polymerase (Taq DNA polymerase)
is used in the reaction which can tolerate the high
temperature of the reaction
...

(a) The process of making recombinant DNA molecule
involves the introduction of a desired gene into the DNA
of a host that will produce the desired protein
...
In
eukaryotic genes the presence of long non-coding introns
may prevent correct expression of these genes in
prokaryotes, which lack RNA-splicing machinery
...
It is known as a recombinant protein
...

The cultures may be used for extracting the desired
protein and then purifying it by using different separation
techniques
...


19
...
This type of culturing method
produces a larger biomass that lead to higher yields of
desired protein
...

HG Khurana discovered DNA ligase enzyme in to phage
in 1969
...
coli in 1972
...

They are present in bacteria to provide a type of defense
mechanism called the 'restriction modification system' and
so called system
...
The first
component includes restriction endounuclease, that
identify the introduced foreign DNA and cut it into ieces,
Same kind of sticky end in different individual molecule
of DNA are also produced by these molecular scissors
...

NEET/AIPMT (2013-2017) Questions

20
...


22
...
Since DNA fragments are negatively
charged molecules they can be separated by forcing them
to move towards the anode under an electric field through
a medium/matrix
...

(d) Alternative selectable markers have been developed
which differentiate recombinant from non-recombinants
on the basis of their ability to produce colour in the
presence of chromogenic substrate
...
This results into inactivation of
the enzyme, which is referred to as insertional
inactivation
...
Presence of insert results into
insertional inactivation of the galactosidase and the
colonies do not produce any colour, these are identified
as recombinant colonies
...


(b)

24
...


(a)

26
...


(b)

28
...


(a)

30
...


(a)

32
...


(c)

34
...


(d)

36
...
The
probe hybridize to single–stranded DNA whose base
sequence allow probe target base-pairing due to
complementary between the probe and target
...
When DNA is placed
into a solution of 100% ethanol, it becomes insoluble
and precipitates out of solution
...
coli or Yeast
...

Now a days PCR and RAPD technique are used for the
characterisation of in vitro clonal propagation in plants
...
They
replicate independently
...

Human genome sequencing is a process that determines
the complete DNA sequence of an organism’s genome at
a single time
...

Commonly used vectors for human genome sequencing
are BAC (Bacterial artificial chromosomes) and YAC
(Yeast artificial chromosomes)
...

A vector is a DNA molecule which is used as a vehicle to
carry the gene of interest to another cell
...

The Taq polymerase enzyme is obtained from Thermus
aquaticus which lives in hot springs
...
Hind II
among these is a type of restriction endonuclease
...

The various stages of processing that occur after the
completion of fermentation or biosynthetic stage which
include separation and purification of product called
downstream processing
...

DNA fragments during gel electrophoresis, separate
(resolve) according to their size due to sieving effect
provided by agarose gel

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