Animal biotechnology
Assignment
Ques-1 Fill in the blanks :
A.Plasmid DNA is treated with alkaline
phosphatase. remove 5’phosphate group from
the linearised DNA.
B. The presence of PUC 18 plasmid in E.Coli provides
Ampicillin resistance to bacteria. C.The insertion of
gene of interest in lac Z gene reading frame leads to
formation white colony, on adding X- Gal to the
medium.
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QUES -2 What are shuttle vectors?
Ans- shuttle vector is a vector that can
propagate in two different host species,
hence, inserted DNA can be tested or
manipulated in two different cell types.
• The main advantage of these vectors is
that they can be manipulated in E. coli
and then used in a system which is more
difficult or slower to use.
• Shuttle vectors can be used in both
eukaryotes and prokaryotes.
• Shuttle vectors are frequently used to
quickly make multiple copies of the gene
in E. coli (amplification).
• One of the most common types of shuttle
vectors is the yeast shuttle vector that
contains components allowing for the
replication and selection in both E. coli
cells and yeast cells.
• The E. coli component of a yeast shuttle
vector includes an origin of replication
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and a selectable marker, such as an
antibiotic resistance like beta lactamase.
• The yeast component of a yeast shuttle
vector
includes
and
autonomous
replicating sequence (ARS), a yeast
centromere
(CEN),
and
a
yeast
selectable marker.O
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Ques - 3 Give Reason:
1.What is the purpose of adding blocking
agent on nitrocellulose membrane during
western blotting?
* Western blot blocking methods are
crucial in order to achieve clean and
reliable results.Protein samples are first
ran on a polyacrylamide gel to separate
proteins by size. The proteins are then
transferred to a nitrocellulose or PVDF
membrane.
* blocking agent is then used to prevent
primary and secondary antibodies from
binding to the membrane non-specifically
(areas without the protein of interest).
blocking methods cover the membrane
surface without interfering with bound
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protein; thus preventing non-specific
antibody binding and non-specific signal
(noise or background) due to the
membrane’s high affinity for proteins.
reliable results.
2.WHY WE USE T4 DNA LIGASE IN GENE
CLONING?
ANS- REASONS ARE:
1.T4 DNA is a ligation enzyme that can be
used to join DNA fragments by catalyzing
the formation of phosphodiester bonds
between juxtaposed 5' phosphate and 3'
hydroxyl termini in double-stranded
DNA using ATP as a coenzyme.
2.Both blunt and cohesive end DNA
ligation, as well as single-stranded nick
repair of DNA, RNA and DNA/RNA, are
possible using the T4 DNA ligase.
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• Applications
• Blunt cohesive end DNA ligation
• Blunt end linker addition
• Single-strand DNA, RNA and DNA/RNA repair
• DNA fragment insertion into vector.
3.Why we use Taq polymerase in PCR?
Ans -Taq polymerase: A thermally stable
DNA polymerase originally isolated from the
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thermophilic bacterium Thermus
aquaticus, which resist inactivation during
denaturation temperatures and allows primer
extension at high temperature.
Ques-4 Restriction enzymes are
called molecular scissors .Illustrate
with example.
1.Restriction enzyme, also called restriction
endonuclease, is a protein produced by bacteria
that cleaves DNA at specific sites along
the molecule.
2. It cleaves DNA into fragments at or near
specific recognition sites within the molecule
known as restriction sites.
3. They have the capacity to recognize specific
base sequences on DNA and then to cut each
strand given place. Hence, they are also called as
‘molecular scissors’.
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Examples of Restriction Enzymes
Enzyme
Obtained from
Recognition Sequence
EcoRI
Escherichia coli
5’GAATTC 3’CTTAAG
EcoRII
Escherichia coli
5’CCWGG 3’GGWCC
BamHI
Bacillus amyloliquefaciens
5’GGATCC 3’CCTAGG
HindIII
Haemophilus influenzae
5’AAGCTT 3’TTCGAA
• One of the most popular restriction
enzymes is called E.coli (bacterium).
• Hundreds of other restriction enzymes
with different sequence
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Ques -5 Compare Calcium Chloride
Method and electroporation – method.
Bacteria which are able to uptake DNA are
called competent which changes the
structure and permeability of the cell
membrane so the naked DNA can enter the
cell.
Calcium chloride transformation
method
• The chemical transformation method
utilizing cacl2 and heat shock to promote
DNA entry into ice cold salt solution
containing cacl2 and heat shock at
42degree celsius.
• This treatment creates transient opening
in the cell wall that enable DNA molecules
to enter the cytoplasm.
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Electroporation
• Competency can also be achieved
through the use of electrical pulses
called electroporation.
• It uses a short pulse of electric charge
to facilitate DNA uptake.It
inducesformations of microscopic
pores within a biological membrane .
• These pores,called electropores ,allow
molecules,ions,and water to pass from
one fide of the membrane to
Ques -6 Describe the following and the
principle behind.
(A)Chain termination method of
sequencing
Chain termination method in which the
sequence of a single stranded DNA
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molecule is determined by enzymatic
synthesis of complementary
polynucleotide chains,these chains
terminating at specific nucleotide
positions.
ChainTerminationMethod
(Sanger Dideoxy Method)
The key principle of the Sanger method was
the use of dideoxynucleotide triphosphates
(ddNTPs) as DNA chain terminators. ... The
DNA bands are then visualized by
autoradiography or UV light, and the
DNA sequence can be directly read off the Xray film or gel image.
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Advantages
1. Purified DNA can be read directly
2. Homopolymeric DNA runs are sequenced
as efficiently as heterogeneous DNA
sequences
3. Can be used to analyze DNA protein
interactions (i.e. footprinting)
4. Can be used to analyze nucleic acid
structure and epigenetic modifications to
DNA
• Limitations
Specific binding of the primer to the
DNA, affecting accurate read-out of the
DNA sequence.DNA structures
affecting the fidelity of the sequence.
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Images of Chain termination
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• Significance of sequencing
1.Information obtained by DNA
sequencing makes it possible to
understand or alter the function of genes.
2.DNA sequence analysis demonstrates
regulatory regions that control gene
expression and genetic “hot spots”
particularly susceptible to mutation.
B. DNA microarray
1.DNA microarrays are solid supports, usually of glass
or silicon, upon which DNA is attached in an organized
pre-determined grid fashion.
2. spot of DNA, called a probe, represents a single
gene.
3.DNA microarrays can analyze the expression of tens
of thousands of genes simultaneously.
4.There are several synonyms of DNA microarrays
such as DNA chips, gene chips, DNA arrays, gene
arrays and biochips.
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• D Figure of DNA Microarray
• Principle
1.The principle of DNA microarrays lies on
the hybridization between the nucleic
acid strands.
2.Using this technology the presence of
one genomic or cDNA sequence in
1,00,000 or more sequences can be
screened in a single hybridization.
3. At least two samples are hybridized to chip.
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Advantages
• Provides data for thousands of
genes in real time.
• Single experiment generates many
results easily.
• Fast and easy to obtain results.
• Promising for discovering cures to
diseases and cancer.
• Different parts of DNA can be used
to study gene expression
Disadvantages
• Expensive to create.
• The production of too many results
at a time requires long time for
analysis, which is quite complex in
nature.
• The DNA chips do not have very
long shelf life.
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C. DNA Fingerprinting
DNA fingerprinting or DNA profiling is a
process used to determine the nucleotide
sequence at a certain part of the DNA that is
unique in all human beings.
🌸The process of DNA fingerprinting was
invented by Sir Alec Jeffrey at the University
of Leicester in 1985.
🌺
Principle
• The area with same sequence of bases
repeated several times is called
repetitive DNA.
• They can be separated as satellite from
the bulk DNA during density gradient
centrifugation
and
hence
called
satellite DNA.
• In satellite DNA, repetition of bases is in
tandem.
The steps involved in DNA
fingerprinting:
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Isolating the DNA.
↓
Digesting the DNA with the help of restriction
endonuclease enzymes.
↓
Separating the digested fragments as per the
fragment size by the process of
electrophoresis.
↓
Blotting the separated fragments onto
synthetic membranes like nylon.
↓
Hybridising the fragments using labelled
VNTR probes.
↓
Analysing the hybrid fragments using
autoradiography.
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• Flow chart representation
Applications
DNA Fingerprinting is used by scientists to
distinguish between individuals of the same
species using only samples of their DNA. It is
a primary method for identifying a individual.
• Forensic Science
• Paternity and Maternity Determination
• Personal Identification and Diagnosis of
Inherited Disorders:
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Ques
-7
Difference
between
Sothernblotting and western blotting ?
Southern Blotting
Western Blotting
A procedure for A blotting procedure
identifying specific used to identify
sequences of DNA. specific amino acid
sequences
in
proteins.
Developed by Edward Developed by Geoge
M.Southern in 1975.
Starks
group
at
Stanford University in
1979
Detects specific DNA Detects
Specific
sequence.
proteins.
Involves Agarose gel Involves SDS PAGE.
electrophoresis
Uses DNA probes
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Uses
primary
and
secondary antibodies.
Ques -8 Discuss the brief Characteristics of
an ideal Cloning Vector.Add a note on
bacterial plasmids and cosmid.
• it must be small in size.
• It must be self-replicating inside host cell.
• It must possess restriction site for Restriction
Endonuclease enzymes.
Introduction of donor DNA fragment must not
interfere with replication property of the vector.
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Bacterial Plasmids
Plasmids are extrachromosomal, double stranded,
autonomously replicating nucleic acid molecules
that are distinct from the chromosome.
• Plasmids can carry few to hundreds of genes. L.
show very little or no homology to host
chromosomal DNA sequences.
• They are not essential and they usually do not
encode essential genes. However, the plasmid
genes may give the bacterium a selective
advantage.
• They replicate independently of chromosome
and are maintained in daughter cells. The
proteins needed for plasmid replication may be
plasmid or chromosomally encoded.
• Viruses are the most common examples of this,
such as herpesviruses, adenoviruses, and
polyomaviruses, but some are plasmids.
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COSMIDS
• Cosmid vector are developed by combining the
featuresof plasmid
vector and
bacteriophage vector.
• The first cosmid vector was described by Collins
in 1978.
• A cosmid is a plasmid that contain phage
sequence that allows the vector to be packaged
and transmitted to bacteria like phage vector
The classic example of cosmid vector is c2RB,
which carries an origin of replication and a cloning
site and has antibiotic-resistant genes. As with the
phage λ vector, the cosmid vector encodes the
cos sequences required for packaging of DNA into
λ capsid.
Ques -9 Explain the process of cDNA library
formation with the help of well labelled diagram.
💐 DNA (cDNA) libraries can also be prepared by isolating mRNAs from tissues which are actively
synthesizing proteins, like roots and leaves in plants,
ovaries or reticulocytes in mammals, etc.The mRNAs are
used for copying them into cDNAs through the use of
reverse transcriptase. Then the cDNA molecule can be
made double stranded and cclone.
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Meaning of cDNA Library:
A cDNA library is defined as a collection of
cDNA fragments, each of which has been
cloned into a separate vector molecule.
Principle of cDNA Library:
In the case of cDNA libraries we produce DNA
copies of the RNA sequences (usually the
mRNA) of an organism and clone them. It is
called a cDNA library because all the DNA in
this library is complementary to mRNA and
are produced by the reverse transcription of
the latter.
The steps involved in the construction
of a cDNA library are as follows:
1. Extraction of mRNA from the eukaryotic Cell:
2. Construction of cDNA from the Extracted
mRNA.
3.Cloning the c-DNA:
A. Linkers
B.Incorporation of restrictions sites.
C.Homopolymer tailing of
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Applications of cDNA Library
1. Discovery of novel genes.
2. Cloning of full-length cDNA molecules for in
vitro study of gene function.
3. Study of the repertoire of mRNAs expressed in
different cells or tissues.
4. Study of alternative splicing in different cells or
tissues.
Advantages of cDNA Library:
A cDNA library has two additional advantages.
First, it is enriched with fragments from actively
transcribed genes. Second, introns do not
interrupt the cloned sequences; introns would
pose a problem when the goal is to produce a
eukaryotic protein in bacteria, because most
bacteria have no means of removing the introns.
Disadvantages of cDNA Library:
The disadvantage of a cDNA library is that it
contains only sequences that are present in mature
mRNA. Introns and any other sequences that are
altered after transcription are not present;
sequences, such as promoters and enhancers, that
are not transcribed into RNA also are not present
in a cDNA library.
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References
1.://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
2.https://microbenotes.com/restriction-enzymerestriction-endonuclease/
3.https://info.gbiosciences.com/blog/western-blotblocking-tips-and-tricks-for-blockingagents?hs_amp=true
4.https://sciencing.com/role-taq-polymerase-pcr7298417.html
5.https://microbenotes.com/dna-sequencing-maxamgilbert-and-sanger-dideoxy-method/
6.https://microbenotes.com/dna-microarray/
7.https://microbenotes.com/dna-fingerprintingprinciple-methods-applications/
8. https://byjus.com/biology/dna-fingerprinting/
9.http://www.biotechnologynotes.com/dnalibraries/notes-on-cdna-library-dna-libraries/517
10.Glick
and
Pasternak
biotechnology book.
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book,Msc
entrance
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