Unit – 1

2 marks –

1. Explain restriction mapping by taking example.

Ans: A restriction map is a map of known restriction sites within a sequence of DNA.
Restriction mapping requires the use of restriction enzymes. In molecular biology,
restriction maps are used as a reference to engineer plasmids or other relatively short
pieces of DNA, and sometimes for longer genomic DNA. There are other ways of
mapping features on DNA for longer length DNA molecules, such as mapping by
transduction.[1]

2. What is DNA polymerase-I larger subunit called as after digesting

with trypsin.
Ans :

3. Explain different chemicals used in disruption of cell membrane

during DNA isolation.

Ans : Ionic detergents, such as SDS, disrupt hydrophobic interactions and hydrogen bonds.
Non ionic detergenet Triton X-100

• Chaotropic agents such as urea and guanidine disrupt hydrogen bonds.

• Reducing agents break disulfide bonds.
• Salts associate with charged groups and at low or moderate

concentrations increase protein solubility.

• Heat disrupts hydrogen bonds and nonpolar interactions.

• Some DNA purification methods incorporate proteases such as proteinase K to digest
proteins.

4. Write about thermostable polymerases used in PCR

reaction?
Ans:PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA
primers designed specifically for the DNA region of interest. In PCR, the reaction is
repeatedly cycled through a series of temperature changes, which allow many copies of the
target region to be produced.

5. Explain different methods used in the identification of purity of

isolated DNA?

• Ans : DNA is polar and therefore insoluble in organic solvents.

• Traditionally, phenol:chloroform (1:1) is used to extract DNA.
• When phenol is mixed with the cell lysate, two phases form. DNA partitions to the
(upper) aqueous phase, denatured proteins partition to the (lower) organic phase.
• DNA is a polar molecule because of the negatively charged phosphate backbone.
• This polarity makes it more soluble in the polar aqueous phase.
• The A260/A280 ratio can be used to determine the purity of a nucleic acid sample.
• Highly purified DNA samples will typically have A260/A280 ratios ranging from 1.8
to 1.9
• Highly purified RNA samples will typically have A260/A280 ratios ranging from 1.9
to 2.0
• An A 260 value of 1.0 indicates the following concentrations:

[dsDNA] = 50 mg/ml; [ssDNA] = 37mg/ml; [ssRNA] = 40 mg/ml

6. Explain mechanism of DNA ligase?

Ans: The mechanism of DNA ligase is to form two covalent
phosphodiester bonds between 3' hydroxyl ends of one nucleotide
("acceptor"), with the 5' phosphate end of another ("donor"). Two ATP
molecules are consumed for each phosphodiester bond formed.

7. Explain general characters of cloning vectors?

Ans: A cloning vector should possess an origin of replication so that it
can self-replicate inside the host cell. It should have a restriction site
for the insertion of the target DNA. It should have a selectable marker
with an antibiotic resistance gene that facilitates screening of the
recombinant organism.

8. What are the general characters of expression vectors?

Ans: Therefore, expression vectors must have the following expression
signals:
•Strong promoter,
•Strong termination codon,
•Adjustment of distance between the promoter and cloned gene,
•Inserted transcription termination sequence, and.
•Portable translation initiation sequence.

9. What are the vectors used for transfer genes into yeast?
Ans: Yeast vectors can be grouped into five general classes, based on
their mode of replication in yeast: YIp, YRp, YCp, YEp, and YLp
plasmids. With the exception of the YLp plasmids (yeast linear
 plasmids), all of these plasmids can be maintained in E. coli as well as
in S.

10. Explain physical and enzymatic method of disruption of bacterial

cell membrane?

Ans :

8 Marks -

1. What are the various steps involved in isolation of phage DNA?

• Ans : Genomic DNA is found in the nucleus of all living cells with the structure of
double-stranded DNA remaining unchanged (helical ribbon).
• The isolation of genomic DNA differs in animals and plant cells. DNA isolation from
plant cells is difficult due to the presence of cell wall, as compared to animal cells.
• The amount and purity of extracted DNA depends on the nature of the cell.
 2. Explain steps involved in isolation and purification of DNA from E. coli.
Ans :
Denaturing agents

• Ionic detergents, such as SDS, disrupt hydrophobic interactions and hydrogen bonds. Non
ionic detergenet Triton X-100

• Chaotropic agents such as urea and guanidine disrupt hydrogen bonds.

• Reducing agents break disulfide bonds.
• Salts associate with charged groups and at low or moderate

concentrations increase protein solubility.

• Heat disrupts hydrogen bonds and nonpolar interactions.

• Some DNA purification methods incorporate proteases such as proteinase K to digest
proteins.

Organic Extraction:

• DNA is polar and therefore insoluble in organic solvents.

• Traditionally, phenol:chloroform (1:1) is used to extract DNA.
• When phenol is mixed with the cell lysate, two phases form. DNA partitions to the
(upper) aqueous phase, denatured proteins partition to the (lower) organic phase.
• DNA is a polar molecule because of the negatively charged phosphate backbone.
• This polarity makes it more soluble in the polar aqueous phase.
• The A260/A280 ratio can be used to determine the purity of a nucleic acid sample.
• Highly purified DNA samples will typically have A260/A280 ratios ranging from 1.8
to 1.9
• Highly purified RNA samples will typically have A260/A280 ratios ranging from 1.9
to 2.0
• An A 260 value of 1.0 indicates the following concentrations:
• [dsDNA] = 50 mg/ml; [ssDNA] = 37mg/ml; [ssRNA] = 40 mg/ml

3. What are the general characters of cloning vectors? Explain plasmids as cloning vector
with an example.

Ans : • Coningvectors

• Used to obtain many copies of cloned DNA

• For preparing genomic DNA Library
• Preparation of probes
• No expression occurs
• These are naturally occurring extra chromosomal double-stranded circular DNA
molecules present in bacteria and yeast.
• These can autonomously replicate inside bacterial cells.
• The Size of plasmids range from about 1.0 kb to over 250 kb.
• The genes required for their own replication (replication proteins) are located very
close to the ori .
• All the other proteins required for replication, e.g. DNA polymerases, DNA ligase,
helicase, etc., are provided by the host cell.
• Thus, only a small region surrounding the ori site is required for replication essential.
 • The other parts present in plasmid can be deleted and foreign sequences can be added
to the plasmid without compromising replication.
• Plasmids are low copy number - 1 to 4 per bacterial cell High copy number - 10 to
1000 per cell
• (Chloremphenicol)
Plasmid constitute 0.5 to 5 % of the total DNA of bacteria
A few bacteria are contain linear plasmids. Eg. Streptomyces sp. And Borella sp.
Plasmids are conjugative and non conjugative
• Plasmids are a. degradative and b. Cryptic plasmids (Will not having apparent
functional coding genes

4. What are the different types of nucleases and polymerases used in rDNA
technology?

Ans : Nucleases

These are the enzymes that degrade DNA molecules by breaking the phosphodiester bonds
that link one nucleotide to the next in a DNA strand.

Based on their site of action these can be broadly Classified into

• Exonucleases: These removes the terminal nucleotide of the DNA molecule by breaking the
phosphodiester bond.

• Endonucleases: These breaks the DNA at internal phosphodiester bond.

Based on the No. of stands on which they act exonucleases are classified as

a) ds DNA: Bal31 is exonuclease isolated from a marine bacterium Alteromonas espejiana is

2+
a Ca dependent enzyme that degrades the nucleotides from both the strands of dsDNA
molecule. Thus it shortens the ds DNA
b) ssDNA: The enzyme isolated from E. coli - exonuclease III digests only one strand of the
dsDNA molecule.

It removes the nucleotide from the 3' terminus of the strand, thus leaving protruding 5'
overhangs. Exonuclease III is used for generating single stranded templates.

S1 nuclease is an endonuclease that is isolated from the fungus Aspergillus oryzae. It is a heat
stable enzyme that functions at high ionic strength, low pH and in the presence of Zn2+ ions.

It cleaves only single stranded DNA. Also, it is able to cleave the single stranded nicks in
dsDNA molecules.

Another type of endonuclease called as DNase I that is isolated from cow’s pancreas is a
non– specific enzymes. It is able to cleave both single and double stranded DNAs.

Polymerases

DNA polymerases are enzymes that catalyse the synthesis of a new DNA strand from a pre-
existing strand.

The enzyme adds deoxyribonucleotides to the free 3’-OH of the chain undergoing elongation.

The direction of synthesis is 5’ to 3’. It has three major requirements for its activity;

• – a template strand for which the enzyme synthesizes a complementary strand;

• – a primer with a free 3’-OH group that hybridizes with the template to form a double

stranded region that initiates the polymerization and

• – a pool of all the four dNTPs that are used to synthesize the new DNA strand.
• Types Polymerases used in GE
• 1. E. coli DNA Polymerase I
• 2. Klenow Fragment
3. Thermostable DNA Polymerase
• 4. Reverse Transciptase
 5. Write about nomenclature and classification of restriction
endonucleases?

Ans : Restriction Endonuclaeases (RE)

• In bacteria, restriction enzymes form a combined system (restriction + modification

system) with modification enzymes that methylate the bacterial DNA.
• Methylation of bacterial DNA at the recognition sequence typically protects the own
DNA of the bacteria from being cleaved by restriction enzyme.
 6. Define restrictions site. How do you estimate approximate number of
restrictions sites present on a given DNA? Types of restrictions sites.
Ans : The restriction enzyme is a protein produced by bacteria that cleaves the
DNA at specific sites. This site is known as the restriction site. The restriction
enzymes protect the live bacteria from bacteriophages. They recognize and cleave
at the restriction sites of the bacteriophage and destroy its DNA.
?????

7. Draw the restriction map for the given fragments: EcoRI digest: 1kb,
2.5kb and 9.5kb; Bam HI digest 2kb, 5kb, 6kb; EcoRI+BamHI digest:
0.5kb, 2kb,5.5kb, 4kb and 1kb.
Ans :

8. What are the characters of Ti and Ri plasmids? How do they are

useful in cloning the genes into plants?
Ans :
 9. Explain different types of topoisomerases and their characters. How
do these are useful in rDNA technology?

Ans :
• 3Types
o 3.1Type I
▪ 3.1.1Type IA
▪ 3.1.2Type IB
▪ 3.1.3Type IC
o 3.2Type II
▪ 3.2.1Type IIA
▪ 3.2.2Type IIB

Types[edit]
DNA topoisomerases are enzymes that have evolved to resolve topological problems in DNA
(Table 2).[10] They do this via transient breakage of one or both strands of DNA. This has led to
the classification of topos into two types: type I, which catalyze reactions involving transient
single-stranded breaks, and type II, which catalyze reactions involving transient double-stranded
breaks (Fig. 3; Table 2). Sub-types exist within these classifications.

Figure 3. Summary of topoisomerase types and catalytic mechanisms. The topoisomerases are
categorized based on whether they catalyze single- (type I) or double-stranded (type II) DNA breaks. The
type I topoisomerases are further subdivided to type IA, IB and IC. Type IA form a transient covalent bond
to the 5ʹ DNA phosphate and function via a strand passage mechanism. Type IB and IC form a transient
covalent bond to the 3ʹ DNA phosphate and function via a controlled-rotation mechanism. Type II
topoisomerases are further subdivided into type IIA and IIB. Both form a transient covalent bond to the 5ʹ
DNA phosphate of both strands of the duplex and function via a strand-passage mechanism.

Type I[edit]
These enzymes catalyze changes in DNA topology via transient single-stranded breaks in DNA.
Reactions can occur on both single- and double-stranded DNA substrates and can proceed via a
'swivel' or 'strand-passage' mechanism (Fig. 3). The range of reactions includes: DNA supercoil
relaxation, unknotting of single-stranded circles, and decatenation, provided at least one partner
has a single-stranded region. In the case of the archaeal enzyme, reverse gyrase, positive
supercoiling of DNA is possible.[24]
Type IA[edit]
Type IA are monomeric and bind to single-stranded segments of DNA. They introduce a
transient single-stranded break through the formation of a tyrosyl-phosphate bond between a
tyrosine in the enzyme and a 5′-phosphate in the DNA. The segment of DNA within which the
break occurs is called the 'gate' or G-segment, and its cleavage allows the passage of another
segment of DNA, the 'transport' or T-segment, to be passed through in a 'strand-passage'
process.[25] This is followed by ligation of the G-segment. For strand passage to occur, topo IA
must undergo a conformational change to open the DNA gate and allow T-segment transfer.
During a DNA relaxation reaction this process changes the linking number of the DNA by +/-1
(Fig. 4). Examples of type IA topoisomerases include prokaryotic topo I and III, eukaryotic topo
IIIα and IIIβ and the archaeal enzyme reverse gyrase. Reverse gyrase, which occurs in
thermophilic archaea, comprises a type IA topo coupled to a helicase, and is the only known
enzyme that can introduce positive supercoils into DNA.[24] The gene encoding reverse gyrase is
also found in some groups of thermophilic bacteria, where it was likely transferred by horizontal
gene transfer from Archaea.[

Role of topoisomerase in DNA replication

Left unresolved, links between replicated DNA will impede cell division. The DNA
topoisomerases prevent and correct these types of topological problems. They do this by
binding to DNA and cutting the sugar-phosphate backbone of either one (type I topoisomerases)
or both (type II topoisomerases) of the DNA strands.

10. What are BACs and YACs? Compare and contrast these vectors
and functions.

Ans :
Unit – 2

2 marks:

1. What do you mean by electroblotting used in Western blotting?

Ans : Electroblotting uses an electric current to pull the negatively charged
proteins from the gel towards the positively charged anode, and into the PVDF
or NC membrane. The proteins move from within the gel onto the membrane while
maintaining the organization they had within the gel.

2. Explain mechanism of construction of recombinant vector.

Ans :

3. What is a TA cloning?
Ans : TA cloning is one of the simplest and most efficient methods for the cloning of
PCR products. The procedure exploits the terminal transferase activity of certain
thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase.

4. What do you mean by a linker and how it is used in cloning of DNA?

Ans : Linker is a chemically synthesized oligonucleotide sequence that is
double-stranded. Linker has two blunt ends. Linker is used to ligate DNA molecules
that have blunt ends to vectors. It contains one or more internal restriction sites.
These restriction sites work as recognition sites for restriction enzymes.

5. What is nitrocellulose membrane and its uses in blotting techniques?

Ans : Nitrocellulose membranes are a popular matrix used in protein blotting
because of their high protein-binding affinity, compatibility with a variety of
detection methods, and the ability to immobilize proteins and glycoproteins.

6. What are the different natural methods of gene transfer?

Ans : The six methods are: (1) Transformation (2) Conjugation (3) Electroporation (4)
Liposome-Mediated Gene Transfer (5) Transduction and (6) Direct Transfer of DNA.

7. If a given eukaryotic gene to be expressed in prokaryote. What is the

promoter to be used and justify your answer?
Ans :
 8. What are dot and slot blots?
Ans : A dot blot (or slot blot) is a technique in molecular biology used to detect
proteins. It represents a simplification of the western blot method, with the exception
that the proteins to be detected are not first separated by electrophoresis.

9. What are the different types of adopters and their applications?

Ans : The 5 adopter categories, in order of their speed of uptake, are:
• Innovators.
• Early Adopters.
• Early Majority.
• Late Majority.
• Laggards.

10. Write about sticky and blunt end cloning of DNA.

Ans : Blunt End Ligation

Endonucleases and exonucleases are the restriction enzymes used in molecular techniques.
They cut the desired DNA portion. Usually, a straight cut creates blunt ends or non-overhanging
ends. These ends can be joined using a DNA ligase enzyme. The joining of two blunt ends is
called blunt end ligation. This does not need matching or complementary ends for ligation.

Sticky End Ligation

Likewise, the restriction enzymes creating a staggered cut leads to two sticky ends or
overhanging ends. The ligation between two overhanging ends with matching or complementary
bases is called sticky end ligation. This sticky end ligation is more efficient than blunt end
ligation. Thus, it is the most desired process in cloning techniques.
8 marks –

1. Write a note on prokaryotic gene expression system.

Ans : The DNA of prokaryotes is organized into a circular chromosome, supercoiled within
the nucleoid region of the cell cytoplasm. Proteins that are needed for a specific function, or
that are involved in the same biochemical pathway, are encoded together in blocks
called operons. For example, all of the genes needed to use lactose as an energy source are
coded next to each other in the lactose (or lac) operon, and transcribed into a single mRNA.

In prokaryotic cells, there are three types of regulatory molecules that can affect the
expression of operons: repressors, activators, and inducers. Repressors and activators are
proteins produced in the cell. Both repressors and activators regulate gene expression by
binding to specific DNA sites adjacent to the genes they control. In general, activators bind
to the promoter site, while repressors bind to operator regions. Repressors prevent
transcription of a gene in response to an external stimulus, whereas activators increase the
transcription of a gene in response to an external stimulus. Inducers are small molecules that
may be produced by the cell or that are in the cell’s environment. Inducers either activate or
repress transcription depending on the needs of the cell and the availability of substrate.

Bacteria such as Escherichia coli need amino acids to survive, and are able to synthesize
many of them. Tryptophan is one such amino acid that E. coli can either ingest from the
environment or synthesize using enzymes that are encoded by five genes. These five genes
are next to each other in what is called the tryptophan (trp) operon((Figure)). The genes are
transcribed into a single mRNA, which is then translated to produce all five enzymes. If
tryptophan is present in the environment, then E. coli does not need to synthesize it and
the trp operon is switched off. However, when tryptophan availability is low, the switch
controlling the operon is turned on, the mRNA is transcribed, the enzyme proteins are
translated, and tryptophan is synthesized.
The tryptophan operon. The five genes that are needed to synthesize tryptophan
in E. coli are located next to each other in the trp operon. When tryptophan is
plentiful, two tryptophan molecules bind the repressor protein at the operator
sequence. This physically blocks the RNA polymerase from transcribing the
tryptophan genes. When tryptophan is absent, the repressor protein does not
bind to the operator and the genes are transcribed.
 2. How are linkers and adopters are used in cloning of a gene?
Ans : What is a Linker?
Linker is a chemically synthesized oligonucleotide sequence that is double-stranded.
Linker has two blunt ends. Linker is used to ligate DNA molecules that have blunt
ends to vectors. It contains one or more internal restriction sites. These restriction
sites work as recognition sites for restriction enzymes.

After ligation, DNA is restricted again with restriction enzymes to produce cohesive
ends. EcoRI-linkers and sal-I linkers are commonly used linkers.

What is an Adaptor?
An Adaptor is a double-stranded oligonucleotide sequence used to link two DNA
molecules together. It is a short sequence with one blunt end and one sticky or
cohesive end. Therefore, it consists of a single-stranded tail at one end, which
enhances the efficiency of DNA ligation.

Moreover, the adaptor has internal restriction sites. Therefore, after ligation, DNA
can be restricted with appropriate restriction enzymes in order to create a new
protruding terminus. One disadvantage of adaptors is that two adaptors can form
dimmers by base pairing with themselves. This can be avoided by treating them
with the enzyme called alkaline phosphatase.

3. What are the different natural gene transfer methods used for bacteria?

Ans :

4. Explain the artificial gene transfer methods used in plants and animals.

Ans : MACRO INJECTION

• Macroinjection is the method tried for artificial DNA transfer to cereals plants that
show inability to regenerate and develop into whole plants from cultured cells.
• Needles used for injecting DNA are with the diameter greater than cell diameter.
• DNA injected with conventional syringe into region of plant which will develop into
floral tillers.
 • Around 0.3 ml of DNA solution is injected at a point above tiller node until several
drops of solution came out from top of young inflorescence.
• Timing of injection is important and should be fourteen days before meiosis.

• This method was found to be successful with rye plants.

• It is also being attempted for other cereals plants.

Microinjection

• Microinjection where the DNA is directly injected into plant protoplasts or cells
(specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometer
diameter) glass needle or micropipette.
• This method of gene transfer is used to introduce DNA into large cells, normally
performed under a specialized optical microscope setup called a micromanipulator.
• The process is frequently used as a vector in genetic engineering and transgenetics to
insert genetic material into a single cell.
• Computerized control of holding pipette, needle, microscope stage and video
technology has improved the efficiency of this technique.

Biolistic Method

• Firstly used by Klein et al (1987) & Sanford et al (1987).

• Also called as,

• Ballistic method / Gene gun method / Particle bombardment / Particle gun method /
Microprojectile

• Genegun is developed to enable penetration of the genetic material containing a gene

of interest in the cell wall .
• 1-2μmtungsten or gold particles are used, coated with the DNA.
 5. Write about natural and artificial gene transfer methods for animals?
Ans : check 3 and 4

6. What is the principle of Southern blotting? Write a note on Southern

blotting and its applications.
Ans :
 7. Describe Northern blotting techniques and how it differs from Southern
blotting?

Ans :

Northern Blot
Southern Blot

Target for
DNA Protein
Detection

DNA extraction, enzymatic digestion by

Sample Prep Protein extraction, protein denatured with SDS
restriction enzymes

Separation Agarose Gel Electrophoresis SDS-PAGE

Membrane Nylon Nitrocellulose or PVDF

Primary antibody(Direct), Primary + Secondary

Nucleic acid probe w/ single stranded
Probe (Indirect) conjugated to fluorophore, reporter
sequence homologous to target DNA
enzyme

Detection
X-ray film, Chemiluminescence CCD Camera, LED or Infrared imaging
Methods
 8. How Western botting is performed and explain its applications?

Ans :
9. How do you express a prokaryotic gene in eukaryote? Explain by taking
an example of Bt cotton?
Ans : ??????

10. Write a note on In situ hybridisation technique and its application.

Ans :
Unit – 3
1. Explain on genomic DNA and plasmid DNA of a given bacteria.
Ans : Genomic DNA constitutes the total genetic information of an organism.
The genomes of almost all organisms are DNA, the only exceptions being some
viruses that have RNA genomes. Genomic DNA molecules are generally large, and
in most organisms are organized into DNA–protein complexes called chromosomes.

A plasmid is a small circular DNA molecule found in bacteria and some other
microscopic organisms. Plasmids are physically separate from chromosomal DNA
and replicate independently.

2. What is cDNA? How it is useful in expression of eukaryotic genes in

prokaryotes?
Ans : In genetics, complementary DNA (cDNA) is DNA synthesized from a single-
stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a
reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to
clone eukaryotic genes in prokaryotes.
3. What is a probe in genetic engineering and its uses?
Ans : A probe is used in screening stage. Probes are always single-stranded, and
can be made of DNA or RNA. If a probe is added to a mixture of different pieces of
DNA (e.g. restriction fragments) it will anneal (base pair) with any lengths of DNA
containing the complementary sequence.
4. Explain on expression screening of DNA library.
Ans :
Library screening is the process of identification of the clones carrying the gene
of interest. Screening relies on a unique property of a clone in a library. The DNA
libraries consist of a collection of probably many thousand clones in the form of
either plaques or colonies on a plate.
5. How size of DNA library can be calculated?

Ans :
6. Explain the role of reverse transcriptase enzyme in preparation of cDNA.
Ans : Reverse transcriptase is used to make a cDNA copy of the mRNA. The
cDNA sample is then amplified by PCR. This yields multiple copies of cDNA without
introns. Reverse transcription followed by PCR allows cloning of genes starting from
the messenger RNA, and thus, identifying the expressed exons of the eukaryotic
gene.
7. Explain the end labelling of the probe DNA.
Ans : End-labeling is a rapid and sensitive method for radioactively, or nonisotopically,
labeling DNA fragments and is useful in visualizing small amounts of DNA. End-labeling can
also be used to label fragments at one end.
8. What are the advantages of next generation sequencing techniques?
Ans : Advantages of NGS include: Higher sensitivity to detect low-frequency
variants. Faster turnaround time for high sample volumes. Comprehensive genomic
coverage.

9. What is a dideoxy nucleotide? How is it used in sequencing?

Ans : Dideoxynucleotides are chain-elongating inhibitors of DNA polymerase,
used in the Sanger method for DNA sequencing. They are also known as 2',3'
because both the 2' and 3' positions on the ribose lack hydroxyl groups, and are
abbreviated as ddNTPs (ddGTP, ddATP, ddTTP and ddCTP).

10. What is a cDNA library?

Ans : A cDNA library is a combination of cloned cDNA (complementary DNA)
fragments inserted into a collection of host cells, which constitute some portion of the
transcriptome of the organism and are stored as a "library".

Unit – 3
1. What is a gene library? Explain construction of genomic DNA library.

• Ans : The term "library" can refer to a population of organisms, each of which carries
a DNA molecule inserted into a cloning vector, or alternatively to the collection of all
of the cloned vector molecules.
• Collection of DNA fragments that have been cloned into vectors so that researchers
can identify and isolate the DNA fragments that interest them for further study.

Construction of genomic DNA Library

1. Isolation of genomic DNA and vector.

2. Cleavage of Genomic DNA and vector by Restriction Endonucleases.
3. Ligation of fragmented DNA with the vector.
4. Transformation of r-DNA in the bacterial cell.
5. Amplification of the r-DNA in bacterial cells.
 2. What is cDNA? Explain synthesis of cDNA. (CO2 and PO2)
Ans : Complementary DNA (cDNA) is a DNA copy of a messenger RNA (mRNA)
molecule produced by reverse transcriptase, a DNA polymerase that can use either DNA or
RNA as a template.
1. Mix RNA sample and primer d(T)23VN in two sterile RNase-free microfuge tubes.
2. Denature RNA for 5 minutes at 70°C. Spin briefly and put promptly on ice. This step is
optional. However, it improves the cDNA yield for long messenger RNAs and GC-rich RNA
regions.
3. Add the following components to one tube.
4.
To the negative control tube, add the following:
5. Incubate the 20 μl cDNA synthesis reaction at 42°C for one hour. If Random Primer Mix
is used, an incubation step at 25°C for 5 min is recommended before the 42°C
incubation.
6. Inactivate the enzyme at 80°C for 5 minutes. Dilute reaction to 50 μl with 30 μl H 2O for
PCR. The cDNA product should be stored at -20°C. For downsteam PCR amplification, the
volume of cDNA product should not exceed 1/10 of the PCR reaction volume.

3. Why is cDNA library is usually constructed for eukaryotes? Explain

construction of cDNA library add a note on its applications.

• Ans : cDNA libraries are very useful for eukaryotic gene analysis.
• Tissue specific

Applications of cDNA library

• Used when reproducing eukaryotic genomes as the amount of information is reduced

to remove the large number of non-coding regions (introns) from the library.
• To express eukaryotic genes in prokaryotes.
• Useful for subsequently isolating the gene that codes for

that mRNA.

• Tissue specific expression profiles

4. Explain screening of library using hybridisation technique.

Ans : Screening of DNA libraries

The process of identifying one particular clone containing the gene of interest from among
the very large number of others in the gene library .
1. Using nucleic acid probe to screen the library based on hybridization with nucleic acids.

2. Analyze the protein product.

Searching the genes of interest in a DNA library

Hybridization to identify the interested DNA or its RNA product

1. Radiolabeled probes which is complementary to a region of the interested gene

Probes:

An oligonucleotide derived from the sequence of a protein product of the gene

A DNA fragment/oligo from a related gene of another species

2. Blotting the DNA or RNA on a membrane

3. Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern)
membrane

5. Explain screening of DNA library by expression and antibody-based

methods

Ans : Expression screening

Identify the protein product of an interested gene

1. Protein activity
2. Western blotting using a specific antibody
If the inserts are cloned into an expression sites, it may be expressed.
Therefore, we can screen for the expressed proteins. However, this screening
may miss the right clone

Example:

Finding the gene for alanine production Grow in alanine deficit medium

Then they are labelled in the petri plate indicates that gene for alanine
production is stored in bacteria

6. Discuss different methods used in preparation of labelled probes.

Ans : There are five basic methods for labeling nucleic acids. These are:

– Nick translation
– Primer extension
– Methods based on RNA polymerase – End labeling methods
– Direct labeling methods

NICK TRANSLATION

• This is done by making single-strand cuts (nicks) in the double

stranded DNA molecule by brief exposure to a dilute solution of an
endonuclease (usually deoxyribonuclease 1 of E. coli).
• DNA polymerase 1 is then used in the presence of at least one
radioactive precursor to “translate” the nick along the molecule in the
5’ to 3’ direction.
• The net result is that a nonradioactive strand of DNA is replaced by a
radioactive strand. The DNA is then denatured and used as a
radioactive probe in hybridization experiments (Southern blots,
Northern blots etc).
• Nick translation can be used with a variety of labels to generate
probes suitable for most hybridization applications. It is also
appropriate for the generation of biotinylated probes.

PRIMER EXTENSION METHOD

• Primers are synthetic oligodeoxyribonucleotides which are

complementary to specific regions of known vector DNA. The 3’
termini of these primers serve as initiation site for template dependent
DNA synthesis by enzymes like DNA polymerase 1.
• DNA polymerase works by extending a short double-stranded region
made by annealing an oligonucleotide primer to the single-stranded
template. Thus this method of uniform labeling requires a primer
 which matches the probe sequence. Radiolabelling of primers can be
done with two methods.
• If the probe sequence is not known then random oligonucleotide
labeling can be used. It is often in the case when natural cellular DNA
is used. These primers are made by adding a mixture of all four bases
at each step in the chemical synthesis reaction.
• The DNA is denatured and the two complementary strands are copied
in the presence of labelled primers as well as nucleoside
triphosphates. The polymerase used is Klenow fragment derived from
DNA polymerase-I of E. coli.

RNA Polymerase based method

• RNA polymerases catalyzes the synthesis of RNA from nucleoside

triphosphates using a DNA template.
• Thus they can incorporate labeled ribonucleotides into RNA during
transcription if such labeled nucleotides are provided to it.
• If a specific site of a vector or DNA is transcribed in such way, RNA
probes (or transcripts) of defined length and sequence can be
obtained.
• Initially RNA probes were obtained from RNA polymerase from E. coli.
This enzyme when used in vitro exhibited very little template and
promoter specificity and therefore produces transcripts which have
been initiated and terminated at random.
• Today, RNA polymerases from a number of phages are used, which
possess a high degree of specificity. Thus transcription can be limited
to sequences cloned downstream of an appropriate promoter.

7. Discuss in detail the Maxam-Gilbert method of DNA sequencing.

 8. Describe automated methods of DNA sequencing.
Ans :

9. Discuss in detail the Sanger’s dideoxy method of DNA sequencing with

an example.
10. What are the different types of Next generation sequencing techniques?
Explain any one of the methods in detail.
 MODULE-4
8M
1. What is a PCR Reaction? Explain in detail the PCR technique.
Ans.
The Polymerase Chain Reaction (PCR) technique, invented in 1985 by Kary B. Mullis,
allowed scientists to make millions of copies of a scarce sample of DNA.
Polymerase chain reaction (PCR) is a laboratory technique used to make multiple copies of a
segment of DNA. PCR is very precise and can be used to amplify, or copy, a specific DNA
target from a mixture of DNA molecules.

A PCR reaction consists of three steps: denaturation, annealing, and extension.

● Denaturation of target (template) – Usually 95oC
● Annealing of primers – Temperature of annealing is dependent on the G+C content –
May be high (no mismatch allowed) or low (allows some mismatch) stringency
● Extension (synthesis) of new strand

PCR Technique:
Assemble a reaction mix containing all components necessary for DNA synthesis.
Subject the reaction mix to an amplification program.
Analyse the product of the PCR reaction (the amplicon)
0.25 mM each primer
0.2 mM each dATP, dCTP, dGTP, dTTP
50 mM KCl
10 mM Tris, pH 8.4
1.5 mM MgCl2
2.5 units polymerase
10^2 - 10^5 copies of template
Total- 50 ml reaction volume

Step 1: Denaturation

As in DNA replication, the two strands in the DNA double helix need to be separated.The
separation happens by raising the temperature of the mixture, causing the hydrogen bonds
between the complementary DNA strands to break. This process is called denaturation.

Step 2: Annealing

Primers bind to the target DNA sequences and initiate polymerisation. This can only occur
once the temperature of the solution has been lowered. One primer binds to each strand.

Step 3: Extension

New strands of DNA are made using the original strands as templates. A DNA polymerase
enzyme joins free DNA nucleotides together. This enzyme is often Taq polymerase, an
enzyme originally isolated from a thermophilic bacteria called Thermus aquaticus.

The order in which the free nucleotides are added is determined by the sequence of
nucleotides in the original (template) DNA strand.The result of one cycle of PCR is two
double-stranded sequences of target DNA, each containing one newly made strand and one
original strand.The cycle is repeated many times (usually 20–30) as most processes using
PCR need large quantities of DNA. It only takes 2–3 hours to get a billion or so copies.

2. What is Real time PCR?

Ans. RTPCR is a Standard PCR with an added probe or dye to generate a fluorescent signal
from the product. Detection of signal in real time allows quantification of starting material.
Performed in specialised thermal cyclers with fluorescent detection systems.

The PCR product grows in an exponential fashion (doubling at each cycle). PCR signal is
observed as an exponential curve with a lag phase, a log phase, a linear phase, and a
stationary phase. The length of the lag phase is inversely proportional to the amount of
starting material.

The first step in a real-time PCR reaction is the conversion of RNA to complementary DNA
(cDNA) – this process is known as reverse transcription.

The next step uses fluorescent reporters and a PCR reaction to amplify and detect specific
genes. Two types of fluorescent reporters are commonly used; these are SYBR green and
Taqman probes.
SYBR green is a dye that fluoresces only when bound to double stranded DNA (i.e the PCR
product)

Taqman probes are made of a gene-specific nucleic acid probe, joined to reporter and
quencher molecules. The probe binds to the DNA between the forward and reverse primer.
While the reporter and quencher are bound to the probe, the quencher absorbs the
fluorescence emitted by the reporter. During the extension phase of the PCR reaction the
probe is degraded, releasing the reporter and allowing its fluorescence to be detected. The
advantage of the Taqman method is that probes with different coloured reporters can be
combined in multiplex assays.

For both SYBR green and Taqman methods, the amount of fluorescence in a sample is
detected in ‘real-time’ and plotted against the cycle number. The amount of fluorescence is
proportional to the amount of PCR product. The time point at which the fluorescence reaches
a defined threshold is relative to the level of gene expression.

3. What is the RAPD technique? Explain in detail the conduction of RAPD and its
applications.

Ans. Randomly amplified polymorphic DNA (RAPD) is a PCR-based technique which uses
arbitrary primers which bind to the nonspecific sites on the DNA and amplify the DNA.
These amplified fragments are then migrated on agarose gel and a difference in the band
pattern is observed. This is a relatively simple and less-expensive technique used for the
diversity study. However, this method has some drawbacks because a very carefully designed
amplification protocol is required for reproducibility of the samples; apart from that, the
DNA template used should be properly purified as the contaminated samples may inhibit the
PCR reaction. RAPD has been successfully used for the identification of antibiotic-resistant
E. coli

Conduction of RAPD:

a. The genomic DNA is first isolated and subjected to PCR amplification.

b. The PCR amplification is done using short stretches of random primers.
c. These primers are about eight to ten nucleotides long and bind to DNA at random
locations.
d. Once the PCR is complete, the PCR amplifiers are separated by agarose gel
electrophoresis.
e. Now if the genomic DNA samples have variation then RAPD gel will show
difference and the band pattern because of mutations if primer fails to bind particular
sites then there will be an absence of particular band.
f. Insertion of few nucleotides will show an increase in band size while the lesion of
new nucleotides will show decrease in band size
Applications of RAPD:

● RAPD have been extensively used for number of horticultural crops in variety
identification, genetic purity and sex determination.
● A specific RAPD marker has been used to select for high and low β-glucan content
between barley varieties.
● RAPD markers are employed in the construction of genetic maps.

4. How do you perform AFLP technique? What are its applications?

Ans. Amplified Fragment Length Polymorphism (AFLP)

• Restriction endonuclease digestion of DNA

• Ligation of adaptors

• Amplification of ligated fragments

• Separation of the amplified fragments via electrophoresis and visualization

• AFLPs have stable amplification and good repeatability

Applications:

AFLP is employed for a variety of applications, such as: to assess genetic diversity within
species or among closely related species, to infer population-level phylogenies and
biogeographic patterns, to generate genetic maps and to determine relatedness among
cultivars.

5.Discuss in detail the DNA fingerprinting technique and its uses.

Ans. The steps involved in DNA fingerprinting technique are:

1. DNA sample: A DNA sample to analyzed is first collected.

2. Extraction of DNA: DNA is then extracted from the sample by lysis method.

3. Digestion by restriction endonucleases: Using a specific restriction enzyme, the DNA

fragment is cut at a specific site. This is done to obtain RFLP (Restricted Fragment Length
Polymorphism).

4. Electrophoresis: Using gel electrophoresis, the DNA fragment of various sizes are
obtained.

5. Denaturing DNA fragment: The gel containing DNA fragments are then immersed in
NaOH solution. This will denature DNA into single-stranded DNA.

6. Southern blotting technique: This is performed to transfer single-stranded DNA from the
gel onto the nitrocellulose membrane.
7. Hybridization: This DNA fragment is subjected to hybridization with a suitable DNA
probe tagged with a radioactive substance.

8. Comparing the sample of the suspect with the evidence: Using X-ray films, the DNA
samples showing radioactivity are compared.

1.Paternity and Maternity

2. Criminal Identification and Forensics

3. Personal Identification

4.Diagnosis of Inherited Disorders

5.Developing Cures for Inherited Disorders

6. Explain different methods of site directed mutagenesis and how they are useful
in rDNA technology?

Ans. Site-directed mutagenesis studies can be extremely useful for elucidating the function of
a gene or protein, or for creating variants of an enzyme with new and improved functions.

There are now many approaches available for generating site-directed mutants, whatever your
purpose. In this post I’ll summarize three techniques that will enable you to produce a wide
range of mutations, and point you to some useful resources to help you get those techniques
up and running.
Note: All of these approaches are good for cloned, genomic or cDNA templates unless
indicated (* means good for cloned templates only, ** means good for gDNA or cDNA
only).

● Technique 1: PCR, with modified primers

Description:

This type of site-directed mutagenesis uses PCR primers designed to contain the desired
change. The PCR primer sequence simply replaces the original sequence – as long as the
changes are minimal enough to allow the primer to anneal to the intended target.

Use for:

Limited base identity changes at the end of the target sequence

5’ or 3’ terminal insertions <100 bases

● Technique 2: Primer extension

Description:

Three Approaches to Site-directed Mutagenesis

Primer extension PCR

Primer extension uses nested primers to mutate a target region. In the diagram, primers B and
C contain the mis-matched sequence to insert bases. The first round of PCR uses primers A-B
and C-D to create two products with the mutated sequence.

The second PCR round is where the smart stuff happens and the new sequence is created.
Since primers B and C contain complementary sequences, the products from the first round
will hybridize after they are denatured following the first PCR cycle. Primers A-D can then
be used to amplify the full-length product that contains the desired mutation. Alterations to
this method can also create deletions or longer additions.

Use for:

Limited, non-random base changes internal to the target sequence

Insertions >100 bases

Deletions < 50 bases

Deletions > 50 bases**

● Technique 3: Inverse PCR

Description:
Inverse PCR is used for mutating plasmids. This method uses two back-to-back primers to
amplify the whole plasmid and the linear product is then ligated back to the circular form.
The primer binding regions can be changed by altering the primer sequences to contain the
desired mutation. Insertions can be made around the primer binding regions by adding
flanking sequences to the primers, and deletions can be made by simply leaving a space
between the two primers.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to

investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and
target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map
specific protein binding sites to RNA.

7. Explain RFLP technique and its applications.

Ans. Restriction Fragment Length Polymorphism (RFLP)

• Genomic DNA digested with Restriction Enzymes

• DNA fragments separated via electrophoresis and transfer to nylon membrane

• Membranes exposed to probes labelled with P32 via southern hybridization

• Film exposed to X-Ray

Applications of RFLP

Can be used for analysis

• Can be used for species or population identification

• Human mt DNA has 2 EcoR1 restriction sites

• Honey bee mt DNA has 5 restriction sites

8. How DNA fingerprinting is different from Southern blotting? Explain technical

differences and applications.

Ans.

Difference:-

A blot is a method of transferring proteins, DNA, or RNA onto a carrier in molecular biology
and genetics. In many cases, this is done after a gel electrophoresis, where the molecules are
transferred from the gel to the blotting membrane, while in other cases, the samples are added
directly to the membrane.

DNA fingerprinting, also known as DNA profile analysis, is based on analysing polymorphic
sections of human DNA using the "Southern" hybridization or southern blotting technique.
Southern blotting is a technique for detecting a specific DNA sequence in a blood or tissue
sample in the laboratory. A restriction enzyme is used to break down a DNA sample into
fragments that can then be separated using gel electrophoresis. The DNA fragments are
transported from the gel to a membrane’s surface. The membrane is subjected to a radioactive
or chemically tagged DNA probe. If the probe binds to the membrane, the sample contains
the probe sequence.

DNA fingerprinting:

1.Paternity and Maternity

2. Criminal Identification and Forensics

3. Personal Identification

4.Diagnosis of Inherited Disorders

5.Developing Cures for Inherited Disorders

Southern Blotting:-
Applications of Southern Blotting

Identifying specific DNA in a DNA sample.

Preparation of RFLP (Restriction Fragment Length Polymorphism) maps

Detection of mutations, deletions or gene rearrangements in DNA

For criminal identification and DNA fingerprinting (VNTR)

Detection and identification of trans gene in transgenic individual

Mapping of restriction sites

For diagnosis of infectious diseases

Prognosis of cancer and prenatal diagnosis of genetic diseases

Determination of the molecular weight of a restriction fragment and to measure relative

amounts in different samples.

9. What are the different variants of PCR? Explain any two variants.

Ans. PCR Modifications

• Nested PCR

• Multiplex PCR

• Tailed primers
• Sequence-specific PCR

• Reverse-transcriptase PCR

• Long-range PCR

• Whole-genome amplification

• RAPD PCR (AP-PCR)

• Quantitative real-time PCR

Nested PCR

• Nested PCR is a modification of PCR that was designed to improve sensitivity and
specificity.

• It involves the use of two primer sets and two successive PCR reactions.

• The first set of primers are designed to anneal to sequences upstream from the second set of
primers and are used in an initial PCR reaction.

Multiplex PCR

– It is the process that amplifies multiple sequences using multiple primers in

thermal cycler.

– It was first described in 1988 to detect deletion in the dystrophin gene – In 2008 it is used
for analysis of microsatellites and SNPs

– In 2020, Real time PCR multiplex assays were designed that combined multiple gene
targets from the Centre (CDC, USA) for Diseases and Control in a single reaction to increase
molecular accessibility and throughput for SARS-C0V2 diagnosis.

– The primer design for all primer pairs has to be optimised so that all primers pairs can work
at same annealing temperature

10. What do you mean by molecular markers? Explain the markers used in DNA
fingerprinting and RFLP.

Ans.

A molecular marker is a molecule contained within a sample taken from an organism

(biological markers) or other matter. It can be used to reveal certain characteristics about the
respective source. DNA, for example, is a molecular marker containing information about
genetic disorders and the evolutionary history of life. Specific regions of the DNA (genetic
markers) are used for diagnosing the autosomal recessive genetic disorder cystic fibrosis,[1]
taxonomic affinity (phylogenetics) and identity (DNA barcoding).
 2M

1. What is nested PCR and its applications?

Ans. Nested PCR is a modification of PCR that was designed to improve sensitivity
and specificity.

• It involves the use of two primer sets and two successive PCR reactions.

• The first set of primers are designed to anneal to sequences upstream from the second set of
primers and are used in an initial PCR reaction.

2. What is a qPCR?

Ans. Quantitative PCR (qPCR)

• PCR product grows in an exponential fashion (doubling at each cycle).

• PCR signal is observed as an exponential curve with a lag phase, a log phase, a linear phase,
and a stationary phase.

• The length of the lag phase is inversely proportional to the amount of starting material.

3. Explain RT-PCR technique?

Ans. RT–PCR is a variation of PCR, or polymerase chain reaction. The two techniques use
the same process except that RT–PCR has an added step of reverse transcription of RNA to
DNA, or RT, to allow for amplification.

4. What do you mean by paternity testing?

Ans. A Paternity Test

• By comparing the DNA profile of a mother and her child it is possible to identify DNA
fragments in the child which are absent from the mother and must therefore have been
inherited from the biological father

5. What is a Molecular beacon and its uses?

Ans. Molecular beacons are stem-loop structures that can be used to detect DNA. A
fluorophore and a quencher are attached to either end of the beacon. Without target, the
fluorescence is quenched but the structure unfolds in the presence of the target and the
fluorescence is increased.
 6. What are the applications of hot start PCR?

AnS. Hot Start PCR allows for reaction set up at room temperature without non-specific
amplification and primer dimer formation. Whereas conventional PCR is often utilized to
make exponential copies of your DNA target sequence without an additional
temperature-sensitive reaction activation component.

7. What is cassette mutation?

Ans. Cassette mutagenesis is a technique for altering a protein sequence at the DNA level by
replacing a section of genetic information with an alternative sequence, normally provided by
a synthetic DNA duplex. First, the gene contained in a suitable vector is cleaved with two
restriction enzymes.

8. How is PCR used in induction of site directed mutation?

Ans. The parent template is removed using a methylation-dependent endonuclease (i.e.

DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the PCR
product). Plasmids are isolated from the resulting colonies, and screened for the desired
modification.

9. If a PCR is conducted for 25 cycles. How many amplified DNA fragments are
produced?

Ans.

10. Explain composition of each PCR cycle.

Ans. A Standard PCR Reaction Mix

0.25 mM each primer

0.2 mM each dATP, dCTP, dGTP, dTTP 50 mM KCl

10 mM Tris, pH 8.4

1.5 mM MgCl2
2.5 units polymerase

102 - 105 copies of template

50 ml reaction volume
 MODULE-5
2M

1. What is a Dicer? Describe its function.

Ans. Dicer plays a pivotal role in the initiation of RNA silencing by recognizing
double-stranded RNAs (dsRNAs) and cleaving them into small RNAs using its RNase
III-like double-stranded RNA-specific nuclease activities. Small RNAs are largely classified
into two groups: small-interfering RNAs (siRNAs) and microRNAs

2. What is a RISC and its function?

Ans. RNA-induced silencing complex, or RISC, is a multiprotein complex that incorporates
one strand of a small interfering RNA (siRNA) or micro RNA (miRNA). RISC uses the
siRNA or miRNA as a template for recognizing complementary mRNA. When it finds a
complementary strand, it activates RNase and cleaves the RNA.

3. What is a siRNA?
Ans. siRNAs are highly specific and usually synthesised to reduce the translation of specific
messenger RNAs (mRNAs). This is done to reduce the synthesis of particular proteins. They
form from double-stranded RNA transcribed and then cut to size in the nucleus before
releasing into the cytoplasm.

4. What is miRNA?
Ans. miRNAs (microRNAs) are short non-coding RNAs that regulate gene expression
post-transcriptionally. They generally bind to the 3'-UTR (untranslated region) of their target
mRNAs and repress protein production by destabilising the mRNA and translational
silencing.

5. What is Flavr Savr tomato?

Ans. The FLAVR SAVR™ tomato was developed through the use of antisense RNA to
regulate the expression of the enzyme polygalacturonase (PG) in ripening tomato fruit. This
enzyme is one of the most abundant proteins in ripe tomato fruit and has long been thought to
be responsible for softening in ripe tomatoes.

6. Applications of antisense RNA.

Ans. Applications of antisense RNA in plants. In plants, antisense RNAs are mainly used in
the inhibition of fruit maturation, virus resistance, flower coloration, starch synthesis, male
sterility, and fertility

7. What is a Humulin?
Ans. Humulin L is an intermediate-acting insulin with a slower onset of action than regular
insulin and a longer duration of activity (up to 24 hours). Due to declining use of
longer-acting insulins, Humulin L was discontinued in 2005.

8. What is the source for gene in preparation of Bt cotton?

Ans. Bt cotton was created through the addition of genes encoding toxin crystals in the Cry
group of endotoxin. When insects attack and eat the cotton plant the Cry toxins or crystal
protein are dissolved due to the high pH level of the insect's stomach.

9. What is TPA? Write its applications.

Ans. Tissue plasminogen activator (tPA) is classified as a serine protease (enzymes that
cleave peptide bonds in proteins). It is thus one of the essential components of the dissolution
of blood clots
A third party administration, also known as a TPA, is an agency that is registered under the
Insurance Regulatory and Development Authority of India (IRDAI). The Third Party
Administrator Licence is required for a company.

10. List the negative aspects of genetic engineering.

Ans. New Allergens in the Food Supply. ...
Antibiotic Resistance. ...
Production of New Toxins. ...
Concentration of Toxic Metals. ...
Enhancement of the Environment for Toxic Fungi. ...
Unknown Harms. ...
Gene Transfer to Wild or Weedy Relatives. ...
Change in Herbicide Use Patterns.

8M
1. What is RNA interference? Explain its applications in rDNA technology.
Ans. RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a
conserved biological response to double-stranded RNA that mediates resistance to both
endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of
protein-coding genes.

applications include -
These include
• Insulin
• Human Growth Hormone
• TissuePlasminogenActivator • Hepatitis-B Vaccine
• Interferons
• MonoclonalAbs
• DNAFingerprinting

INSULIN
• Human Insulin was the first recombinant- derived product, used for the treatment of
individuals suffering from diabetes.
• Insulin is a protein, which is naturally obtained from β-cells of the Islets of Langerhans of
the Pancreas.
• It is a molecule with 51 amino acids in two polypeptide chains (A-chain = 21 and B- chain
= 30 ), held together by disulphide cross linking.
2. How do the recombinant human insulin is produced?
Ans. INSULIN
• Human Insulin was the first recombinant- derived product, used for the treatment of
individuals suffering from diabetes.
• Insulin is a protein, which is naturally obtained from β-cells of the Islets of Langerhans of
the Pancreas.
• It is a molecule with 51 amino acids in two polypeptide chains (A-chain = 21 and B- chain
= 30 ), held together by disulphide cross linking.
3. Discuss the production of genetically modified Bt Cotton.
Ans. Bt cotton has been genetically modified by the insertion of one or more genes from a
common soil bacterium, Bacillus thuringiensis. These genes encode for the production of
insecticidal proteins, and thus, genetically transformed plants produce one or more toxins as
they grow. The genes that have been inserted into cotton produce toxins that are limited in
activity almost exclusively to caterpillar pests (Lepidoptera). However, other strains of
Bacillus thuringiensis have genes that encode for toxins with insecticidal activity on some
beetles (Coleoptera) and flies (Diptera). Some of these genes are being used to control pests
in other crops, such as corn.
Resistance and resistance management
It seems likely that some cotton pests could develop resistance to Bt crops if they are
extensively used, and this has already been documented for some species. For example,
bollworm have developed some resistance to the Cry1Ac, Cry1F, and Cry2A Bt toxins. One
primary resistance management strategy is having a refuge, either non-Bt cotton or other
non-Bt crops or wild hosts that serve as a source of susceptible insects that would potentially
breed with any resistant insects generated in Bt cotton fields. The offspring of this mating
would be susceptible to Bt toxins (assuming the genetic trait for resistance is at least partially
recessive). A second resistance management tactic is the introduction of Bt crops that
produce two or more relatively dissimilar toxins. Presumably, it is less likely that any one
insect will be simultaneously resistant to more than one toxin. Current Bt cotton varieties
express two or three Bt toxins.
4. Explain the limitations and negative aspects of genetic engineering?
Ans.
1. The nutritional value of foods can be less.
When animals grow, and mature quickly, the nutritional value of that product can be reduced.
This can be seen in poultry products today with the white striping that is found in meat
products. That striping is a fat deposit that was created, often in the breast meat, because of
the rapid growth of the bird. In chickens, Good Housekeeping reports that this can increase
the fat content of the meat consumed by over 220%. At the same time, the amount of protein
that is received is also reduced.

2. Pathogens adapt to the new genetic profiles.

Genetic engineering can create a natural resistance against certain pathogens for plants and
animals, but the natural evolutionary process is geared toward creating pathways. Bacteria
and viruses evolve a resistance to the resistance that is created by the genetic engineering
efforts. This causes the pathogens to become stronger and more resistant than they normally
would be, potentially creating future health concerns that are unforeseen.

3. There can be negative side effects that are unexpected.

Genetic engineering is guaranteed to make a change. Many of those changes are positive,
creating more and healthier foods. Some of those changes, however, can be negative and
unexpected. Making a plant become more tolerant to drought might also make that plant
become less tolerant to direct sunlight. Animals may be modified to produce more milk, but
have a shortened lifespan at the same time so farmers suffer a greater livestock.
4. The amount of diversity developed can be less favourable.
At some point, genetically engineered plants and animals make it “into the wild” and interact
with domestic species. This results in a crossing of “natural” and “artificial” organisms. The
engineered organisms often dominate, resulting in only a modified species over several
generations, reducing the diversity that is available.
5. Copyrighted genetic engineering can have costly consequences.
Many companies copyright their genetic engineering processes or products to maintain their
profitability. If a farmer plants genetically modified crops and the pollination process causes
another farmer in the field to have those modified crops grow, there have been precedents for
legal actions against the “unauthorised” farmer. This can have several costly consequences,
from fewer farmers wanting to work to a higher cost for the seeds that are planted.

6. This knowledge and technology can be easily abused.

At the moment, genetic engineering in humans is being used to treat specific disorders that
threaten the health or wellbeing of individuals. In time, the approach in humans could be like
what is already being done with plants and animals. Genetic engineering can change specific
traits, which could create human outcomes that are ethically questionable or easily abused.

The advantages and disadvantages of genetic engineering show that the results can be
generally positive, but there must be controls in place to manage the negative when it occurs.

5. Discuss various applications of rDNA technology in agriculture.

Ans.

6. Explain the various applications rDNA technology in Medicine.

Ans. [11:48 AM, 5/27/2022] Nirmal: Hormones
• Human Insulin Genes Have Been Implanted In Bacteria Which,
17 May 2022
therefore, become capable of synthesising insulin. Bacterial insulin is identical to human
insulin, since it is coded by human genes. Diabetics have been receiving bacterial insulin in
test programmes, and it appears to be
as effective as insulin from animal sources.
***Growth hormone
[11:48 AM, 5/27/2022] Nirmal: Vaccines
• Gardasil or Silgard is a vaccine proven to prevent certain types of human papillomavirus
(HPV) specifically HPV types 16, 18, 6, and 11
***Vaccines against malaria and hepatitis may also be produced in the near future.
[11:48 AM, 5/27/2022] Nirmal: Interferons-
• Interferons are virus induced proteins having antiviral in action.
**Interferon provides natural defence against such viral diseases as hepatitis and influenza. It
also appears to be effective against certain types of cancer, especially cancer of the breast and
lymph nodes.
[11:49 AM, 5/27/2022] Nirmal: Sickle cell disease
• The specific molecular defect was identified as a single amino acid substitution of valine for
glutamic acid at position 6 of the beta-globin polypeptide chain. genetic mutation in the
globin gene as a change in the codon GAG to GTG.
[11:49 AM, 5/27/2022] Nirmal: Cystic Fibrosis -
• Thereareseveralmechanismsmutationscausedproblemswith the cystic fibrosis
transmembrane conductance regulator [CFTR ]protein.
• ΔF508,for instance,creates protein
• that doesn't fold
• population result in proteins that are too short because production
it ended prematurely.
• thatdonotuseenergynormally,donotallowchloride,iodide
and thiocyanate to cross the membrane appropriately, or are degraded at a faster rate than
normal.

7. Discuss applications of rDNA technology in environmental management?

Ans. Using genetic engineering techniques one can transform microorganisms such as
• bacteria or yeast, or
• transform cells from multicellular organisms such as insects
or mammals
• with a gene coding for a useful protein, such as an enzyme,
so that the transformed organism will overexpress the desired protein.
One can manufacture mass quantities of the protein by growing the transformed organism in
bioreactor equipment using techniques of industrial fermentation, and then purifying the
protein.
Some genes do not work well in bacteria, so yeast, insect cells, or mammalian cells, each a
eukaryote, can also be used.

•
These techniques are used to produce
medicines:
• insulin,
• human growth hormone,
• vaccines,
• supplements such as tryptophan, aid in the production of food
(chymosin in cheese making) and fuels.
Other applications involving genetically engineered bacteria being investigated: making the
bacteria perform tasks outside their natural cycle:
1. Makingbiofuels,
2. Cleaning up oil spills,
3. Carbon And Other Toxic Waste[and
4. Detecting Arsenic In Drinking Water.
8. Explain design and application of siRNA.
Ans. siRNA is a synthetic RNA duplex designed to specifically target a particular mRNA for
degradation. While siRNA provides the opportunity to induce gene knockdown in a variety of
cell lines, their utility is limited to cells that are amenable to transfection of synthetic
oligonucleotides.

Since siRNAs achieve transient silencing, experiments are limited to relatively short time
frames on the order of 2-4 days. siRNAs can also be used for knockdown of non-protein
coding genes, such as long noncoding RNAs (lncRNA).
siRNA function

The success of RNAi experiments depends on the efficiency of gene knockdown. Early work
on siRNA design established conventional guidelines for siRNA structural attributes that led
to reasonable functional knockdown in specific cases1.

The properties of potent siRNAs were further refined by performing large-scale functional
studies that defined thermodynamic and sequence-based rules for rational siRNA design2.
These design algorithms greatly improved the reliability of identifying potent siRNA
sequences. The Dharmacon SMARTselection algorithm was the first comprehensive rational
design strategy applied to commercial collections. While research is continually striving to
identify molecules with greater activity and specificity, siRNAs designed by SMARTselection
strategies, such as siGENOME and ON-TARGETplus reagents, remain the mo mst effective
reagents on the market

Applications

siRNAs are widely used to assess the individual contributions of genes to an assortment of
cellular phenotypes including cytokinesis11, apoptosis12, insulin signalling 13,14 and cell
differentiation15.

siRNA screens have been used to identify novel pathways16 and have had significant impact
in validating targets for a number of cellular processes and diseases including cancer17,18,
HIV infection19 and hepatitis20. Finally, in vivo RNAi has been used for target validation
studies in animal disease models and has the potential to be used for therapeutic purposes
where disease-causing genes are selectively targeted and suppressed

9. Discuss applications of rDNA technology in Industry.

Ans.
1. It allows for a faster growth rate.
Genetic engineering allows plants or animals to be modified so their maturity can occur at a
quicker pace. Engineering can allow this maturity to occur outside of the normal growth
conditions that are favourable without genetic changes as well. Even if there are higher levels
of heat or lower levels of light, it becomes possible to expand what can be grown in those
conditions.

2. It can create an extended life.

Genetic modification can help to create resistance to common forms of organism death. Pest
resistance can be included into the genetic profiles of plants so they can mature as a crop
without any further additives. Animals can have their genetic profiles modified to reduce the
risks of common health concerns that may affect the breed or species. This creates the
potential for an extended lifespan for each organism.

3. Specific traits can be developed.

Plants and animals can have specific traits developed through genetic engineering that can
make them more attractive to use or consumption. Different colors can be created to produce
a wider range of produce. Animals can be modified to produce more milk, grow more muscle
tissue, or produce different coats so that a wider range of fabrics can be created.
4. New products can be created.
With genetic engineering, new products can be created by adding or combining different
profiles together. One example of this is to take a specific product, such as a potato, and alter
its profile so that it can produce more nutrients per kcal than without the genetic engineering.
This makes it possible for more people to get what they need nutritionally, even if their food
access is limited, and this could potentially reduce global food insecurity.
5. Greater yields can be produced.
Genetic engineering can also change the traits of plants or animals so that they produce
greater yields per plant. More fruits can be produced per tree, which creates a greater food
supply and more profits for a farmer. It also creates the potential for using modified
organisms in multiple ways because there is a greater yield available. Modified corn, for
example, can be used for specific purposes, such as animal feed, ethanol, or larger cobs for
human consumption.

6. Risks to the local water supply are reduced.

Because farmers and growers do not need to apply as many pesticides or herbicides to their
croplands due to genetic engineering, fewer applications to the soil need to occur. This
protects the local watershed and reduces the risk of an adverse event occurring without
risking the yield and profitability that is needed.

7. It is a scientific practice that has been in place for millennia.

Humans in the past may not have been able to directly modify the DNA of a plant or animal
in a laboratory, but they still practised genetic engineering through selective breeding and
cross-species or cross-breeding. People would identify specific traits, seek out other plants or
animals that had similar traits, and then breed them together to create a specific result.
Genetic engineering just speeds up this process and can predict an outcome with greater
regularity.
10. What are the various achievements of genetic engineering?
Ans.