11.

BIOTECHNOLOGY: PRINCIPLES & PROCESSES

· Traditional concept of Biotechnology-deals with the of usage of live organisms or their enzymes
for products & processes useful to humans. Eg:- microbe-mediated processes of making curd, bread
or wine.
. Modern molecular biotechnology- refers to the use of genetically modified organisms for large
scale production of products. Biotechnology deals with:
- Microbe-mediated processes (making curd, bread, wine etc).
- In vitro fertilization (test-tube baby programme).
- Synthesis and using of a gene.
- Preparation of DNA vaccine.
- Correcting a defective gene.
· 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’.
PRINCIPLES OF BIOTECHNOLOGY
Core techniques of modern biotechnology
· Genetic engineering: The technique in which genetic material (DNA & RNA) is chemically altered
and introduced into host organisms to change it’s phenotype.
· Bioprocess engineering: Maintenance of sterile ambience in chemical engineering processes for
growing only desired microbe/eukaryotic cell on a large scale.

Basis for principles: 1.variations and unique combinations of genetic setup in sexual reproduction,
some of which may be beneficial and advantageous to the organism as well as the population
compared to preserved genetic information of Asexual reproduction.
2. Isolation and introduction of only one or a set of desirable genes into the target organism when
compared to Traditional hybridisation procedures used in plant and animal breeding.

Techniques of genetic engineering:

→ creation of recombinant DNA-a new combination of circular, self-replicating DNA created in vitro
is known as recombinant DNA.
· The process of joining and inserting a foreign piece of DNA into a host organism to produce new
genetic combinations is called recombinant DNA technology.
→ use of gene cloning-Cloning: making multiple identical copies of any template DNA.
Clones: multiple identical copies of any template DNA.
→gene transfer
·First recombinant DNA (rDNA) was produced by Stanley Cohen & Herbert Boyer (1972).
· They isolated an antibiotic resistance gene (piece of DNA) from a plasmid of Salmonella
typhimurium. It was linked with a plasmid vector and transferred into E. coli. As a result, the gene
was expressed & multiplied in E. coli.
→ Vector: Plasmid DNA that is used to deliver/ transfer an alien piece of DNA into the host
organism.
→ Plasmid: self- replicating, circular, extra-chromosomal DNA.
Basic steps in genetically modifying an organism
a) Identification of DNA with desirable genes: Traditional hybridisation leads to inclusion and
multiplication of undesirable genes along with desired genes. In genetic engineering, only desirable
genes are introduced.
b) Introduction of the identified DNA into the host: A vector DNA such as plasmid is used to deliver
an alien piece of DNA into the host organism.
c) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny: A piece of
alien DNA has no sequence called Origin of replication (ori) needed to start replication. So, it cannot
multiply itself in host organism. Hence alien DNA is integrated into the vector DNA (that has ori). It
then, multiplies and inherits along with host DNA.
→ ORI: a specific DNA sequence called the origin of replication, which is responsible for initiating
replication.
TOOLS OF RECOMBINANT DNA TECHNOLOGY:
Key tools include
→ Enzymes-restriction enzymes, polymerase enzymes, ligases
→Cloning Vectors and
→Competent host organism
→ ENZYMES
1. Restriction Enzymes (‘molecular scissors’)
- The enzymes that cut DNA at specific sites into fragments.
- They belong to a class of enzymes called nucleases.
- In 1963, two enzymes responsible for restricting growth of bacteriophage in E. coli were isolated.
One enzyme added methyl groups to DNA. The other (restriction endonuclease) cut DNA.
- More than 900 restriction enzymes have been isolated from over 230 strains of bacteria.
Naming of the restriction enzymes:
- First letter indicates genus name. The second two letters indicate species name of prokaryotic cell
from which they were isolated.
E.g. EcoR I comes from E. coli RY 13 (R = the strain. Roman number /s = the order in which the
enzymes were isolated from that strain of bacteria)
Types of Restriction enzymes:
· Exonucleases: They remove nucleotides from the ends of the DNA.
· Endonucleases:- They cut at specific positions within the DNA. E.g. EcoR I.
- They bind to specific recognition sequence of the DNA and cut the two strands at specific points.
- The first restriction endonuclease is Hind II isolated from Haemophilus influenzae during 1968
and was characterised five years later. It cuts DNA molecules by recognizing a specific sequence of 6
base pairs. This is called the recognition sequence for Hind II.
Functioning of REN
-REN inspects, binds and cut each of the two strands of the double helix at specific point of
recognition sequence, along the length of the DNA in their sugar-phosphate back bone.
- Restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA. It is
a sequence of base pairs that read the same on the two strands in 5' → 3' direction and in 3' →
5' direction. E.g. Palindromic nucleotide sequence for EcoR I is
5' —— GAATTC —— 3'

3' —— CTTAAG —— 5'

- Restriction enzymes cut the strand a little away from the centre of the palindrome sites, but
between the same two bases on the opposite strands. This leaves single stranded overhanging
stretches at the ends. They are called sticky ends. They form H-bonds with their complementary cut
counterparts. Hence REN are also called “Molecular scissors” .This stickiness facilitates action of the
enzyme DNA ligase.

- When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of
sticky-ends and these are joined together by DNA ligases.

2. Polymerase enzymes-These make multiple identical copies of any template DNA.

3. Ligases- These enzymes join together , the two cut ends of DNA. Hence are also called “ Molecular
Stitchers” .

→ CLONING VECTOR
- It is a carrier DNA, that transfers a foreign DNA attached to it and self-replicate inside the host
cells.
Uses of Vectors: *They help easy linking of foreign DNA.
*Selection of Recombinants from Non-recombinants.
Types of Vectors:
- Plasmids are self-replicating circular extra-chromosomal DNA of bacteria. Some plasmids have
only 1-2 copies per cell. Others have 15-100 or even more copies per cell.
- Bacteriophages -have very high copy numbers of their genome per cell /within the bacterial cells.
-When the cloning vectors are multiplied in the host, the linked piece of DNA is also multiplied to the
numbers equal to the copy number of the vectors.
- Vectors for cloning genes in plants & animals
· Modified tumor inducing (Ti) plasmid of Agrobacterium tumefaciens is used to deliver genes of
interest into a variety of plants via infective mode.
· Disarmed Retroviral infection deliver desirable genes into animal cells. Retroviruses in animals can
transform normal cells into cancerous cells.
Features required for cloning into a vector

a. Origin of replication (ori)

- This is a sequence where replication starts.
-A piece of DNA linked to ori can replicate within the host cells. This also controls the copy number
of linked DNA. So, for getting many copies of the target DNA, it should be cloned in a vector whose
origin support high copy number.

b. Selectable marker (marker gene)

- It is a gene that helps to identify and eliminate the non-transformants and selectively permitting
the growth of only transformants.
- Transformation- Is a procedure through which a piece of DNA is introduced in a host bacterium.
Such bacterium is transformant. If transformation does not take place, it is called as a non-
transformant.
-Selectable markers of E. coli include the genes encoding resistance to antibiotics like ampicillin,
chloramphenicol, tetracycline, kanamycin etc. Normal E. coli cells have no resistance against these
antibiotics.
- But this type of selection of recombinants is a difficult procedure because it needs simultaneous
plating on 2 plates having different antibiotics.
So, alternative selectable markers have been developed Eg., -galactosidase enzyme coding gene
based on their ability to produce colour in presence of a chromogenic substrate. Insertional
inactivation of this gene leads to colourless Recombinant colonies in response to Chromogenic
substrate (X-gal) and helps to differentiate them from blue coloured Non-recombinant colonies.

c. Cloning sites
- These are the recognition sites for restriction enzymes.
-To link the alien DNA, the vector preferably needs a single recognition site.
- MCS, a multiple cloning site -More than one recognition sites for a particular enzyme generate
several fragments. It complicates the gene cloning.

- Ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic
resistance genes.
E.g. In vector pBR322, foreign DNA is ligated at Bam
H I site of tetracycline resistance gene. As a result,
recombinant plasmid is formed. If ligation does not
occur, it is called non-recombinant plasmid.

Structure of pBR322- It was developed by two

Mexican creators Boliver and Rodriguez in 1972.
It includes the following
· Restriction sites(Cloning sites/the recognition
sites): - These are the recognition sites for
restriction enzymes, one recognition site for a
particular enzyme.Eg., Hind III, EcoR I, BamH I, Sal I,
Pvu II, Pst I, Cla I.
· ori: a sequence where replication starts.
· Selectable marker (marker gene): Antibiotic resistance genes: ampR and tetR.
· Rop: codes for the proteins involved in the replication of plasmid.

IMPORTANT NOTE- Inactivation of a gene encoding an enzyme due to insertion of foreign DNA is
called insertional inactivation. Here, the recombinant plasmids lose tetracycline resistance due to
insertion of foreign DNA.
When the plasmids are introduced into E. coli cells, 3 types of cells are obtained:
*Non-transformants: They have no plasmid. So they are not resistant to either tetracycline or
ampicillin.
*Transformants with non-recombinant plasmid: They are resistant to both tetracycline & ampicillin.

*Transformants with recombinant plasmid: They are resistant only to ampicillin.

- Recombinant plasmids can be selected out from non-recombinant ones by plating transformants
on ampicillin medium.

-Then the transformants are transferred on tetracycline medium.

- The recombinants grow in ampicillin medium but not on tetracycline medium. But, non-
recombinants grow on the medium containing both the antibiotics.
- Thus, one antibiotic resistance gene helps to select the transformants. The inactivated antibiotic
resistance gene helps to select recombinants.
- But this type of selection of recombinants is a difficult procedure because it needs simultaneous
plating on 2 plates having different antibiotics.
So, alternative selectable markers have developed based on their ability to produce colour in
presence of a chromogenic substrate.
 - In this, a recombinant DNA is inserted into
the coding sequence (gene) of an enzyme,
-galactosidase. So, the gene is inactivated
(insertional inactivation). Such colonies do not
produce any colour. These are identified as
recombinant colonies.
- If the plasmid in bacteria have no insert, it
gives blue coloured colonies in presence of
chromogenic substrate.
- Whereas a Non-transformant doesn’t grow
at all.

3. Competent Host (For Transformation with

Recombinant DNA)
- Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. So bacterial cells are
made ‘competent’ to take up alien DNA or plasmid as follows:
- Treat bacterial cells with a specific concentration of a divalent cation (e.g. calcium),which increases
the efficiency with which rDNA enters the bacterium through pores in cell wall.
→rDNA is then forced into such cells by Incubating on ice.
→ Place them briefly at 420C (heat shock).
→ Put them back on ice.
Other methods to introduce alien DNA into host cells

Micro-injection method Biolistics (gene gun) method

· Micro-injection: In this, recombinant DNA is directly injected into the nucleus of an animal cell.

· Biolistics (gene gun): In this, cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA. This method is suitable for plants.
· ‘Disarmed pathogen’ vectors: They infect the cell and transfer the recombinant DNA into the host.
E.g. Disarmed A. tumefaciens and Retroviruses deliver desirable genes into plant and animal cells
respectively.
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
It includes -isolation of DNA, fragmentation of DNA by REN & isolation of a desired DNA fragment,
Amplification of Gene of Interest using PCR, ligation of the DNA fragment into a vector & transferring
the recombinant DNA into the host, culturing the host cells in a medium at large scale and extraction
of the desired product and downstream processing.
1.Isolation of the Genetic Material (DNA)
- Treat the bacterial cells/plant or animal tissue with enzymes like lysozyme (bacteria),
cellulase (plants), chitinase (fungus) etc. The cell is broken releasing DNA and other
macromolecules (RNA, proteins, polysaccharides and lipids).
- RNA is removed by treating with ribonuclease. Proteins are removed by treatment with
protease. Other molecules are removed by appropriate treatments.

DNA that separates out can be removed by spooling

- When chilled ethanol is added, purified DNA precipitates out as a collection of fine threads
in the suspension which can be removed by Spooling.
2. Cutting of DNA at Specific Locations - Purified DNA is incubated with the specific
restriction enzyme at the optimal conditions.
3.Separation of desired length of DNA fragments and isolation: The DNA fragments are
separated by a technique called gel electrophoresis.
Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.
DNA is negatively charged. So it moves towards the anode. DNA fragments are separated according
to their size through sieving effect of the agarose gel (a polymer extracted from sea weeds). The
smaller sized fragment moves farther.
*The process is repeated with the vector DNA also.
*DNA fragments can be seen as bright orange coloured bands when they are stained with
ethidium bromide and exposed to UV radiation.
*DNA bands are cut out from agarose gel. It is called elution. The cut-out gene of interest
and cut vector are mixed and ligase is added. It creates recombinant DNA.
4. Amplification of Gene of Interest using PCR- Polymerase Chain Reaction (PCR) is the
synthesis of multiple copies of the gene of interest in vitro using 2 sets of primers & the
enzyme DNA polymerase.
- Primers are small chemically synthesized oligonucleotides that are complementary to the
regions of DNA.

Steps of PCR:
*Denaturation: It is the heating of target DNA (gene of interest) at high temperature (940 C)
to separate the strands. Each strand acts as template for DNA synthesis.
*Annealing: It is the joining of the two primers (at 520 C) at the 3’ end of the DNA
templates.
*Extension: It is the addition of nucleotides to the primer at 720C using a thermostable DNA
polymerase called Taq polymerase. It is isolated from a bacterium, Thermus aquaticus. It
remains active in high temperature during the denaturation of double stranded DNA.
Through continuous replication, the DNA segment is amplified up to 1 billion copies.The
amplified fragment can be used to ligate with a vector for further cloning.
5.Ligation of the DNA fragment (Gene of interest/foreign DNA/Alien DNA) to a Vector: The
Vector DNA and the Gene of interest are incubated with enzyme Ligases. This results in
rDNA molecule.
6. Insertion of Recombinant DNA into Host Cell- The rDNA is introduced into recipient
(host) cell / organism by increasing it’s competency. They take up DNA from its surrounding.
7.Identifying and eliminating Non-transformants- If a recombinant DNA bearing ampicillin
resistant gene is transferred into E. coli cells, the host cells become ampicillin-resistant cells.
- If the transformed cells are spread on agar plates containing ampicillin, only transformants
will grow. Untransformed recipient cells will die.
8. Obtaining the Foreign Gene Product- The aim of recombinant DNA technology is to
produce a desirable protein.
*If a protein encoding foreign gene is expressed in a heterologous host, it is called a
recombinant protein.
* The cells with foreign genes can be grown in laboratory. The cultures are used to extract
the desired protein and purify it by using separation techniques.
*The cells can also be multiplied in a continuous culture system. Here, the used medium is
drained out from one side while fresh medium is added from the other. It maintains the
cells physiologically active log/exponential phase and so produces a larger biomass. It yields
more desired protein.

Bioreactors- These are the vessels in which raw materials are biologically converted to
specific products, enzymes etc., using microbial, plant, animal or human cells.
-Bioreactors are used to produce large quantities of products. They can process 100-1000
litres of culture.
- A bioreactor provides the optimal growth conditions (pH,temperature, substrate, salts,
vitamins, oxygen) to get desired product.
Structure of a Simple stirred tank bioreactor: The most commonly used bioreactors are of
stirring type (stirred-tank bioreactor). It is usually cylindrical or with a curved base to
facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen
availability. Alternatively, air can be bubbled through the reactor.
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).
9. Downstream Processing- It is a series of processes such as separation and purification
of products after the biosynthetic stage.
- The product is formulated with suitable preservatives.
- Such formulation undergoes thorough clinical trials as in case of drugs and
- Strict quality control testing.
 Previous solved Model papers and question papers
PART –A
MCQs
1.Plant cells are bombarded with high velocity micro particles of gold or tungsten coated
with DNA in a method known as. (Main-2023)
(a)Microinjection (b)Biolistics (c)Heat shock (d)Vector mediated
Ans: (b)Biolistics

2.The commonly used vector for cloning genes in animals is (Main-2023)

(a)Retro virus (b)Disarmed retro virus
(c)Disarmed Ti-plasmid (d)Agrobacterium tumefaciens
Ans: (b)Disarmed retro virus

3.Enzymes which remove nucleotides from the ends of the DNA are (Supp-2023)
(a)Endonucleases (b)Exonucleases (c)DNA ligases (d)DNA polymerases
Ans: (b)Exonucleases

4.Gel electrophoresis is used to. (2nd Supp-2023)

(a)Clone the gene (b)Separation of DNA fragments
(c)Cut the DNA fragments (d)Join the DNA fragments
Ans: (b)Separation of DNA fragments

5.Which of the following statements are correct for the enzyme Taq polymerase?
(i)Taq polymerase is thermally unstable
(ii)It requires primers for carrying out the process of polymerisation
(iii)Taq polymerase is thermally stable
Choose the correct option(A1-2024)
(a) i and ii (b) i and iii (c) ii and iii (d) i , ii and iii
Ans: (c) ii and iii

(1Mark each)
1.” Gel electrophoresis is considered as a very important technique in rDNA technology”.
Why?
Ans. The DNA fragments separate (resolve) according to their size / length through
the sieving effect provided by the Agarose gel.

2.Name the technique used to check the progression of REN and to separate the DNA
fragments.
Ans. Gel electrophoresis.

3.Define polymerase chain reaction.(March-2014) /

What is polymerase chain reaction.(July-2014)/(Main-2021)
Ans. Polymerase chain reaction Is a technique of amplifying(multiplying) a DNA
fragment in to approximately a billion copies within a short time in-vitro.
4.Retroviruses are disarmed before using to deliver desirable genes in to animal cells.
Why?(July-2014)
Ans. Retroviruses are disarmed in order to avoid the transformation of normal cells
in to cancerous cells.

5.Name the molecular scissors used in rDNA technology. (March-2016) / Why restriction
enzyme is called molecular scissors? (Sup-2020)
Ans. REN (Restriction endonucleases)

6.What are plasmids? (July-2016) / (Sup-2020)/ (Main-2022)/(Supp-2022)

Ans. Plasmids are autonomously replicating circular extra-chromosomal DNA of
bacteria.

7.What is Micro-Injection? (March-2017)

Ans. Micro-Injection is a method to introduce recombinant DNA directly by
injecting into the nucleus of an animal cell.

8.Name the plasmid present in Agrobacterium tumefaciens. (March-2019)

Ans. Ti- plasmid

9.Name the stain used to visualise DNA fragments in gel electrophoresis. (July-
2019)/(Main-2023)/A3-2024
Ans. Ethidium bromide
PART –B (2Marks each)
1.Explain the method of introduction of Alien DNA into bacterial cells.
Ans. Bacterial cells are made ‘competent’ to take up alien DNA or plasmid as follows:
→ Treat bacterial cells with a specific concentration of a divalent cation (e.g. calcium)
→ DNA enters the bacterium through pores in cell wall
→ Incubate the cells with recombinant DNA on ice
→ Place them briefly at 420C (heat shock)
→ Put them back on ice
→ Bacteria take up recombinant DNA.
2.Draw a neat labelled diagram of a Stirred tank bioreactor.(March-2014) /
Draw a neat labelled diagram of a simple stirred tank bioreactor. (3-Marks, July-2016 /
March-2017)
3. Mention two techniques to make Host competent in r-DNA technology. (July-2016)
/(Main-2021)
Write the methods to introduce alien DNA into host cells. (March-2018) /
Mention any two methods of introducing alien DNA into host cells. (July-2019)/(Supp-
2022)/ (Main-2023)
Ans. →Heat shock → Micro-injection
→ Biolistics or (gene gun) → Disarmed pathogen vectors
(Any two)
4.Draw a neat labelled diagram of a typical agarose gel electrophoresis. (March-2016)/
With a neat labelled diagram, explain the technique of gel electrophoresis in recombinant
DNA technology.(Main-2022)-5Marks
Ans.

→DNA fragments are separated by a technique called gel electrophoresis.

→ DNA being negatively charged is forced to move towards the anode under an electric
field in matrix (agarose gel).
→ DNA fragments are separated according to their size through sieving effect of the agarose
gel hence the smaller sized fragments move farther.
→ Ethidium bromide- is used to stain the separated DNA fragments and visualise as bright
orange coloured bands when exposed to UV radiation.
→Elution- The separated bands of DNA are cut out from the agarose gel and extracted from
the gel piece.

5.What is the significance of selectable marker in cloning vector? (March-2017) /

Mention the importance of selectable markers in plasmids. (July-2018)
Ans. Selectable markers help in identifying and eliminating non-transformants
and selectively permitting the growth of the transformants.

6. Write the restriction site of EcoRI enzyme. (1-Mark,March-2018) /

What are palindromic nucleotide sequences? Write the restriction site of Eco RI enzyme.
(July-2018)
Ans. Palindromic nucleotide sequences in the DNA refer to a sequence of base pairs
that read the same on the two strands in 5' → 3' direction and in 3' → 5' direction.
 Restriction site of Eco RI enzyme 5' —— GAATTC —— 3‘
3' —— CTTAAG —— 5‘
7.With an example, explain the convention for naming restriction endonucleases
scientifically. (March-2019)/(A2-2024)
Ans. The convention for naming restriction endonucleases scientifically is the first
letter indicates genus. The second two letters indicate species of prokaryotic cell from which
they were isolated.
E.g. EcoRI comes from Escherichia coli
The first capital letter E indicates genus Escherichia.
The second two letters co indicate species coli .
The second capital letter R denotes the strain RY 13.
Roman number I = the order in which the enzyme was isolated from that strain of bacteria.
PART –C (3Marks each)
1.Mention any three features of Vectors that are most suitable for the purpose of rDNA
technology. / Explain the features of Cloning vectors.(March-2015)
Ans. a. Origin of replication (ori)- This is a sequence where replication starts and for
getting many copies of the target DNA, it should be cloned in a vector whose origin support
high copy number.
b. Selectable marker (marker gene) - It is a gene that helps to select the transformants and
eliminate the non-transformants and also selectively permits the growth of the
Transformants.
-Selectable markers of E. coli include the genes encoding resistance to antibiotics like
ampicillin, chloramphenicol, tetracycline, kanamycin etc.
c. Cloning sites-These are the recognition sites for restriction enzymes to link the alien DNA.
The vector needs a single or very few recognition or MCS, a multiple cloning site inside a
scorable marker. E.g. In vector pBR322, foreign DNA is ligated at Bam H I site of tetracycline
resistance gene.
2.Mention the different steps of process of rDNA technology.(March-2014)
Ans. →Isolation of the Genetic Material (DNA)
→fragmentation of DNA by restriction endonucleases
→isolation of a desired DNA fragment
→ ligation of the DNA fragment into a vector
→ transferring the recombinant DNA into the host
→ culturing the host cells in a medium at large scale
→ extraction of the desired gene product
→Down stream processing of the products as finished product ,ready for
marketing
3. Explain in brief, the separation and isolation of DNA fragments.
Ans. →DNA fragments are separated by a technique called gel electrophoresis.
→ DNA being negatively charged is forced to move towards the anode under an electric
field in matrix (agarose gel ).
→ DNA fragments are separated according to their size through sieving effect of the agarose
gel hence the smaller sized fragments move farther.
→ Ethidium bromide- is used to stain the separated DNA fragments and visualise as bright
orange coloured bands when exposed to UV radiation.
→Elution- The separated bands of DNA are cut out from the agarose gel and extracted from
the gel piece.
4.Draw a labelled diagram of plasmid pBR322.(July-2015) / (July-2017) / (March-2018)
Ans.

5.Write the important use of the following in r-DNA technology (July-2018)

a)PCR- is the synthesis of multiple copies of the gene of interest in vitro using 2 sets of
primers & the enzyme DNA polymerase.
b)Ethidium bromide- is used to stain the separated DNA fragments and visualise as bright
orange coloured bands when exposed to UV radiation.
c)Bioreactor- is used to produce large quantities of products where large volumes (100-
1000 litres) of culture can be processed.

6.a)Mention four tools required for rDNA technology.(2-Marks) / Write any four tools
used in rDNA technology.(2-Marks, July-2019)/(Main-2023)
b)With reference to gel electrophoresis, what is Elution? (1-Mark)(March-2019) / (1-Mark,
Sup-2020)
Ans. Tools required for rDNA technology are:
→ Enzymes-Restriction enzyme, DNA ligases and DNA polymerases.
→ Vector DNA.
→ Competent host.
→ Bioreactors.
Elution- The separated bands of DNA are cut out from the agarose gel and extracted from
the gel piece.
7.Explain the process of denaturation , annealing and extension with respect to
polymerase chain reaction. (Sup-2020)
Ans. Steps of PCR:
*Denaturation: It is the heating of target DNA (gene of interest) at high temperature (940 C)
to separate the strands. Each strand acts as template for DNA synthesis.
*Annealing: It is the joining of the two primers (at 520 C) at the 3’ end of the DNA
templates.
*Extension: It is the addition of nucleotides to the primer at 720C using a thermostable DNA
polymerase called Taq polymerase. It is isolated from a bacterium, Thermus aquaticus. It
remains active in high temperature during the denaturation of double stranded DNA.
Through continuous replication, the DNA segment is amplified up to 1 billion copies.The
amplified fragment can be used to ligate with a vector for further cloning.

8.What are endonucleases? Write the rules for naming the restriction enzymes.(Main-
2021)
Ans. Endonucleases:-enzymes that cut at specific positions within the DNA.
Convention for naming restriction endonucleases scientifically- first letter indicates genus.
The second two letters indicate species of prokaryotic cell from which they were isolated.
E.g. EcoRI comes from Escherichia coli
The first capital letter E indicates genus Escherichia.
The second two letters co indicate species coli .
The second capital letter R denotes the strain RY 13.
Roman number I = the order in which the enzyme was isolated from that strain of bacteria.

PART –D (5Marks each)

1.a)Explain how DNA is isolated from cells. (3)
b)Differentiate between Endonuclease and Exonuclease.(1) / (2-Marks, March-2020)/
Mention two classes of nucleases and suggest their respective roles. (2) /
Classify restriction enzymes. Mention the function of each.(July-2015)/(A1-2024)
c)What is the uniqueness of Taq polymerase? (1)
Ans. a)Isolation of the Genetic Material (DNA)
- Treat the bacterial cells/plant or animal tissue with enzymes like lysozyme (bacteria),
cellulase (plants), chitinase (fungus) etc. The cell is broken releasing DNA & other
macromolecules (RNA, proteins, polysaccharides & lipids).
- RNA is removed by treating with ribonuclease. Proteins are removed by treatment with
protease. Other molecules are removed by appropriate treatments.
-When chilled ethanol is added, purified DNA precipitates out as a collection of fine threads
in the suspension which can be removed by Spooling and is called as Elution.
b)· Exonucleases:- They remove nucleotides from the ends of the DNA.
· Endonucleases:-They cut at specific positions within the DNA.
c)Taq polymerase is a thermo stable DNA , which remain active during the high
temperature.
2.What is polymerase chain reaction? Name the bacterium from which the polymerase
enzyme used in this technique is obtained. Write the schematic representation of this
technique./ Name the process by which a desired gene amplification can be done. Explain
the process briefly. / Write the use of PCR and write the scientific name of the bacterium
from which the thermostable DNA polymerase enzyme is obtained.(Main-2022) (Supp-
2022)
Ans. Polymerase Chain Reaction (PCR) is the synthesis of multiple copies of
the gene of interest in vitro using 2 sets of primers (small chemically synthesized
oligonucleotides that are complementary to the regions of DNA) & the enzyme DNA
polymerase.
Taq polymerase enzyme is isolated from a bacterium, Thermus aquaticus.

Schematic representation of Polymerase Chain Reaction (PCR)

Steps of PCR:
*Denaturation: It is the heating of target DNA (gene of interest) at high temperature (940 C)
to separate the strands. Each strand acts as template for DNA synthesis.
*Annealing: It is the joining of the two primers (at 520 C) at the 3’ end of the DNA
templates.
*Extension: It is the addition of nucleotides to the primer at 720C using a thermostable DNA
polymerase called Taq polymerase. It is isolated from a bacterium, Thermus aquaticus. It
remains active in high temperature during the denaturation of double stranded DNA.
Through continuous replication, the DNA segment is amplified up to 1 billion copies.The
amplified fragment can be used to ligate with a vector for further cloning.
3.Write diagrammatic representation of rDNA technology. / Diagrammatically represent
rDNA technology. (3-Marks, March-2020) / (A3-2024)
b)Write a note on down stream processing.(2-Marks, July-2014) / Supp-2023
What is down stream processing? (1-Mark, July-2017)/ (A3-2024)
Ans. a)
(b)Down stream Processing-It is a series of processes such as separation and purification of
products after the biosynthetic stage before it is ready for marketing as a finished product.
- The product is formulated with suitable preservatives.
- Such formulation undergoes thorough clinical trials as in case of drugs
- Strict quality control testing.
4.Read the following statements and write one appropriate term for each (A1-2024)
(a)Autonomously replicating, circular, extra chromosomal DNA- Plasmid.
(b)Method to introduce rDNA in animal cells- Micro-injection/ Disarmed retro viral
infection
(c) Method to introduce rDNA in plant cells- Biolistics or (gene gun)/ Modified Ti-plasmid
vector introduction via Agrobacterium tumefaciens infection mode.
(d)Specific DNA sequence responsible for initiating replication- Ori sequence
(e)Enzyme used to form the DNA fragment- DNA polymerase
5.Give reasons for the following statements. (A2-2024)
(a)Alien DNA is linked with the Ori site of a vector in gene cloning.
To replicate and control the copy number of alien DNA within the host cells.
(b)Restriction enzymes are called “molecular scissors”.
They cut DNA at specific sites into fragments.
(c)DNA ligase can be called “molecular glue”.
These enzymes join together the two cut ends of DNA.
(d)Gel electrophoresis is considered as a very important technique in rDNA technology.
DNA fragments are separated according to their size through sieving effect of the agarose
gel.
(e)DNA fragments move towards anode under electric field through a medium in gel
electrophoresis.
DNA being negatively charged is forced to move towards the anode under an electric field in
matrix (agarose gel ).