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BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
DEPARTMENT OF BIOTECHNOLOGY
Faculty Name : Ms. K. Ramya Faculty Code : HTS 1277 Subject Name : Genetic Engineering and Genomics Subject Code : BT6603 Year & Semester : III & VI
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
DEPARTMENT OF BIOTECHNOLOGY COURSE DETAILS
Faculty Name : Ms. K. Ramya Faculty Code: HTS 1277
Subject Name: Genetic Engineering and Genomics Subject Code: BT6603
Department: Biotechnology Year & Semester: III & VI
COURSE OUTCOMES
On completion of this course, the students will be able to
CO No Course Outcomes Knowledge Level
C315.1 Understand about the cloning of commercially important genes and production of
recombinant proteins K2
C315.2 Understand about the construction and screening of DNA libraries K2
C315.3 Discuss about the gene and genome sequencing techniques K2
C315.4 Explain about the microarrays, analysis of gene expression and proteomics K2
C315.5 Understandarticulate the applications of genome analysis and genomics K2
Mapping of Course Outcomes with Program Outcomes and Program Specific Outcomes
BT6602 PO1 PO2 PO3 PO4 PO5 PO6 PO7 PO8 PO9 PO10 PO11 PO12 PSO1 PSO2 PSO3
PSO4
C315.1 2 - - - - - - - - - - 3 - - - -
C315.2 2 - - - - - - - - - - - - - - -
C315.3 2 - - - - - - - - - - - - - - -
C315.4 2 - - - - 3 3 - - - - 3 - - - -
C315.5 1 - - - - 3 3 - - - - 3 - - - -
BT6602 PO1 PO2 PO3 PO4 PO5 PO6 PO7 PO8 PO9 PO10 PO11 PO12 PSO1 PSO2 PSO3
PSO4
C315 2 - - - - 3 3 - - - - 3 - - 3 -
Mapping Relevancy
1: Slight (Low) 2: Moderate (Medium) 3 Substantial (High) - : No correlation
K1 – Remember; K2 – Understand; K3 – Apply; K4 – Analyze; K5 – Evaluate; K6 - Create
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
BT6603 GENETIC ENGINEERING AND GENOMICS
UNIT-I
PART A
1. State four safety guidelines in creating rDNA.
i Care should be taken that novel organism created should not be normal
ii. Treatment with alkaline phosphatase to increase the number of recombinants
iii. Appropriate insert and vector size should be selected.
iv. Proper technique for increasing the transformation efficiency should be
selected.
2. What are the basic properties of a plasmid vector? (MAY 2014)
Low molecular weight, ability to confer readily selectable phenotypic trait on host
cells, single sites for a large number of restriction endonuclease preferably in
genes with a readily scorable phentotype.
3. What are DNA modifying enzymes? Give one example.(DEC ‘2010) (May’
2012,2016)
These are the enzymes that are involved in the degradation synthesis and
alteration of nucleic acids.DNA ligase - An enzyme which seals single stranded
nicks between adjacent nucleotides in a duplex DNA chain.
Alkaline phosphatase - Removes the 5’ phosphates and replaces it by hydroxyl
group.
4. What are isoschizomers and Neoschizomers? Give any two examples.(DEC’2013,
2015)
Isoschizomers are the enzymes obtained from different sources but recognizes the
same target.Ex-SmaI ,XmaI.
Neoschizomers are restriction enzymes that recognize the same nucleotide
sequence as their prototype but cleave at a different site.
For example:Prototype MaeII A^CGT produces DNA fragments with a 2-base 5'
extension Neoschizomer TaiI ACGT^ produces DNA fragments with a 4-base 3'
extension
5. If you add ligase to alkaline phosphates treated vector does the ligation takes place?
Justify your answer.
No. because the phosphate group at 5 end is replaced by hydroxyl group, so
phosphodiester linkage is not formed.
6. Define restriction endonuclease.(MAY 2013,2014), (Dec' 2016)
Nuclease that recognizes specific nucleotide sequences in a DNA molecule and
cleaves or nicks the DNA particular site.
7. State the difference between 3 types of restriction endonuclease
Type I – recognizes and cleaves the DNA upto 1000 bp away from the site.
Type II – recognizes and cleaves at specific target site.
Type III- recognizes and cleaves at 26-30 bp away from the target size.
8. Give few examples for restriction endonuclease. (MAY 2013), (Dec' 2016). Sma I, Hae III, Hind III, Bam HI.
9. Difference between cohesive sticky ends & blunt end.
S.NO STICKY END BLUNT END
Cut he bases around the center
of symmetry
Cut he bases at the
center of symmetry
2. Promotes effective ligation Ligation efficiency is
less
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
3.
Eg.GAATTC → G
AATTC
CTTAAG CTTAA
G
GAATTC → GAA
TTC
CTTAAG CTT
AAG
10. Give the steps involved in creating a recombinant plasmid.
Isolation of gene of interest, selection of vector, cutting vector with restriction enzyme,
joining with DNA ligase, transformation, screening and expression of the cloned genes.
11. Draw a neat diagram of plasmid vector.
12. Which type of restriction enzyme is used for creating rDNA? (May’ 2012)
Justify?
Type II restriction enzyme because they produce cleavage at or near host
specificity site.
13. What are Biotin and Avidin? What is their role in rDNA technology? (DEC’ 2010).
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds,
reptiles and amphibians deposited in the whites of their eggs. Biotin, historically
known as Vitamin H is a water-soluble B-complex vitamin (vitamin B7)
discovered by Bateman in 1916. It is composed of a ureido
(tetrahydroimidizalone) ring fused with a tetrahydrothiophene ring. A valeric acid
substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring.
Biotin is a coenzyme in the metabolism of fatty acids and leucine, and it plays a
role in gluconeogenesis. Their role in rDNA technology is for tagging purpose.
14. What is RCGM and mention its role (MAY’ 2010)
Review Committee on Genetic Modification (India). Monitors research projects
safety aspects
15. Differentiate a promoter and an enhancer (MAY’ 2011), (Dec' 2016)
Enhancer DNA sequences bind transcription factors called enhancer-binding
proteins which increase the rate of transcription. Enhancer sequences may be
kilobases away from the gene they influence. An enhancer complex may interact
with promoter complexes by bringing the sites into direct contact. Promoter a
regulatory region of DNA located upstream of a gene, providing a control point
for regulated gene transcription
16. Give a name of a modifying enzyme that helps in converting blunt end DNA to
sticky
ends (MAY’ 2011, 2012)
Terminal transferase
17. What is meant by expression vector? (May’ 2012), (Dec' 2016) Plasmids or phages carrying promoter regions to cause expression of inserted
DNA sequences
18. Define Cosmid.(MAY 2013), (Dec' 2016). A vector designed to allow cloning of large segments of foreign DNA. They are
hybrids
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
composed of the COS sites of lambda inserted into a plasmid.
Helps in joining the 2 DNA fragments.
19. What are the requirements for an efficient prokaryotic expression vector?
(DEC’2013), (Dec' 2016) Constructing the optimal promoter, optimizing translation initiation, maintenance
of
the stability of mRNA, effect of codon size ,transcription termination, plasmid
copy
number ,plasmid stability and host cell physiology.
20. Differentiate adaptors from linkers.(DEC’ 2010), (MAY’ 2010, 2014))
Linkers are short stretches of double stranded DNA of length 8-14 bp and have
recognition site for 3-8 RE. These linkers are ligated to blunt end DNA by ligase.
Adapters are linkers with cohesive ends or a linker digested with RE, before
ligation. The most widely used definition is cut linkers also called as adapters.
21. What are phagemids? How are they different from cosmids? (DEC’ 2010)
A phagemid or phasmid is a type of cloning vector developed as a hybrid of the
filamentous phage M13 and plasmids to produce a vector that can grow as a
plasmid, and
also be packaged as single stranded DNA in viral particles. Phagemids contain an
origin of replication (ori) for double stranded replication, as well as an f1 ori to
enable single
stranded replication and packaging into phage particles. Many commonly used
plasmids
contain an f1 ori and are thus phagemids. Phagemids: F1 origin cloned into a
plasmid
Cosmids: Cos sites cloned into a plasmid
22. Name any two eukaryotic Transcription factors. Give their functions. (MAY 2013),
(Dec' 2016) General transcription factors of the pre-initiation complex are required for the
expression of all structural genes transcribed by RNA polymerase II (Ex): TFIID
→ TBP + TFIIA, TFIIB, TFIIF, TFIIE,TFIIH
Specific transcription factors bind to proximal promoter DNA sequences or distal
enhancer elements. (Ex): homeodomain proteins, p53, etc
23. Name any two special features of pBR 322. (MAY 2013) pBR322 is 4361 base pairs in length and contains the replicon of plasmid pMB1,
the ampR gene, encoding the ampicillin resistance protein (source plasmid
RSF2124) and the tetR gene, encoding the tetracycline resistance protein (source
plasmid pSC101). The plasmid has unique restriction sites for more than forty
restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites
for restriction enzymes HindIII and ClaI within the promoter of the tetR gene.
There are 6 key restriction sites inside the ampR gene. The origin of replication or
ori site in this plasmid is pMB1 (a close relative of ColE1).
24. What are the applications of polylinkers. (Dec 2014)
A multiple cloning site (MCS), also called a polylinker, is a short segment of
DNA which contains many (up to ~20) restriction sites - a standard feature of
engineered plasmids.
Restriction sites within an MCS are typically unique, occurring only once within a
given plasmid. MCSs are commonly used during procedures involving molecular
cloning or subcloning. Extremely useful in biotechnology, bioengineering, and
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
molecular genetics, MCSs let a molecular biologist insert a piece of DNA or
several pieces of DNA into the region of the MCS. This can be used to create
transgenic organisms, also known as genetically modified organisms (GMOs).
25. Write briefly about genetic makeup of T4 phage. (May 2015).
PART –B
1. Define the following terms Recombinant DNA, Recombinant DNA technology,
Recombinant protein & Recombinant RNA and explain in detail about the steps
involved in creating a rDNA molecule
Recombinant DNA: The result of combining DNA fragments from different sources.
Recombinant DNA technology: A set of techniques which enable one to manipulate DNA.
One of the main techniques is DNA cloning (because it produces an unlimited number of
copies of a particular DNA segment), and the result is sometimes called a DNA clone or gene
clone (if the segment is a gene), or simply a clone. An organism manipulated using
recombinant DNA techniques is called a genetically modified organism (GMO).
Recombinant protein: A protein whose amino acid sequence is encoded by a cloned gene.
Recombinant RNA: A term used to describe RNA molecules joined in vitro by T4 RNA
ligase.
STEPS INVOLVED IN CREATION OF A rDNA
1. Selection of the gene of interest
2. Selection of of vector
3. Treatment with restriction endonuclease
4. Ligation using ligase
5. Transformation in suitable host
6. Screening of the recombinant plasmid
7. Expression of particular protein.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
2. i. Differentiate between selectable marker and reporter gene.
ii. Give examples of selectable markers in bacteria, yeast, plant, insect and
mammalian systems Selectable marker: A gene whose expression allows the identification of:
1. A specific trait or gene in an organism.
2. Cells that have been transformed or transfected with a vector containing the marker gene.
Give examples for the marker genes and also explain in detail about the insertional
inactivation mechanism.
Reporter gene: A gene that encodes a product that can readily be assayed. Thus reporter genes are used to
determinate whether a particular DNA construct has been successfully introduced into a cell,
organ or tissue.
A number of marker genes are, described as screenable genes or scoreable genes or reporter
-Glucuronidase(GUS) and
luciferase(lux) genes.
Selectable markers in bacteria All antibiotic resistance eg.Ampicillin.Tetracycline,kanamycin,chloramphenicol etc.
Selectable markers in yeast URA 3 ,LEU 2,TRP 1
Selectable markers in plant Neomycin phosphotransferase (npt II), Hygromycin phosphotransferase (hpt
II),dihydrofolate reductase
Selectable markers in insect Polyhedrin gene
Selectable markers in mammalian systems Antibiotic resistance eg.Neomycin
3. Write short notes on the following modifying enzymes. (MAY’ 2011,
2014,2016,2017), (DEC’ 2015) i. Exonucleases ii.Alkaline phosphatases iii.Terminal transferases
iv.Methylases. v. Taq polymerase vi. DNA Ligase
i. Exonucleases (4)
Exonucleases are enzymes (found as individual enzymes, or as parts of larger enzyme
complexes) that cleave nucleotides one at a time from an end of a polynucleotide chain.
These enzymes hydrolyze phosphodiester bonds from either the 3' or 5' terminus of a
polynucleotide molecule.
ii. Alkaline phosphatases (4)
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for removing phosphate
groups from many types of molecules, including nucleotides, proteins, and alkaloids. The
process of removing the phosphate group is called dephosphorylation. As the name suggests,
alkaline phosphatases are most effective in an alkaline environment.in gene cloning it is used
to prevent circularisation of the vector DNA molecule.
iii. Terminal transferases (4)
Terminal transferase catalyzes the addition of nucleotides to the 3' terminus of DNA.
Interestingly, it works on single-stranded DNA, including 3' overhangs of double-stranded DNA,
and is thus an example of a DNA polymerase that does not require a primer. It can also add
homopolymers of ribonucleotides to the 3' end of DNA. The much preferred substrate for this
enzyme is protruding 3' ends, but it will also, less efficiently, add nucleotides to blunt and 3'-
recessed ends of DNA fragments. Cobalt is a necessary cofactor for activity of this enzyme.
Terminal transferase is useful for at least two procedures:
Labeling the 3' ends of DNA: Most commonly, the substrate for this reaction is a fragment of
DNA generated by digestion with a restriction enzyme that leaves a 3' overhang, but
oligodeoxynucleotides can also be used. When such DNA is incubated with tagged nucleotides
and terminal transferase, a string of the tagged nucleotides will be added to the 3' overhang or to
the 3' end of the oligonucleotide.
Adding complementary homopolymeric tails to DNA: This clever procedure was
commonly used in the past to clone cDNAs into plasmid vectors, but has largely
been replaced by other, much more efficient techniques. The principles of this
technique are depicted in the figure below. Basically, terminal transferase is used to
tail a linearized plasmid vector with G's and the cDNA with C's. When incubated
together, the compementary G's and C's anneal to "insert" the cDNA into the vector,
which is then transformed into E. coli.
Terminal transferase is a mammalian enzyme, expressed in lymphocytes. The
enzyme purchased commercially is usually produced by expression of the bovine
gene in E. coli.
iv. Methylases. (4)
A methylase is an enzyme that attaches a methyl group to a molecule. There are methylases
that can methylate DNA, RNA, proteins, or small molecules, for example, DNA
methyltransferase, which methylates cytosine residues and adenine residues in DNA.in gene
manipulation its used to prevent action of restriction activity.
v. Taq polymerase
Taq polymerase is a thermostable DNA polymerase named after the thermophilic
bacterium Thermus aquaticus from which it was originally isolated by Chien et al. in
1976. Its name is often abbreviated to Taq Pol or simply Taq. It is frequently used in the
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short
segments of DNA.T. aquaticus is a bacterium that lives in hot springs and hydrothermal
vents, and Taq polymerase was identified as an enzyme able to withstand the protein-
denaturing conditions (high temperature) required during PCR. Therefore, it replaced the
DNA polymerase from E. coli originally used in PCR.[3] Taq's optimum temperature for
activity is 75–80 °C, with a half-life of greater than 2 hours at 92.5 °C, 40 minutes at 95
°C and 9 minutes at 97.5 °C, and can replicate a 1000 base pair strand of DNA in less
than 10 seconds at 72 °C. One of Taq's drawbacks is its lack of 3' to 5' exonuclease
proofreading activity[4] resulting in relatively low replication fidelity. Originally its error
rate was measured at about 1 in 9,000 nucleotides. The remaining two domains act in
coordination, via coupled domain motion. Some thermostable DNA polymerases have
been isolated from other thermophilic bacteria and archaea, such as Pfu DNA
polymerase, possessing a proofreading activity, and are being used instead of (or in
combination with) Taq for high-fidelity amplification.Taq makes DNA products that
have A (adenine) overhangs at their 3' ends. This may be useful in TA cloning, whereby a
cloning vector (such as a plasmid) that has a T (thymine) 3' overhang is used, which
complements with the A overhang of the PCR product, thus enabling ligation of the PCR
product into the plasmid vector.
vi. DNA Ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, (EC 6.5.1.1) that
facilitates the joining of DNA strands together by catalyzing the formation of a
phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in
living organisms, but some forms (such as DNA ligase IV) may specifically repair double-
strand breaks (i.e. a break in both complementary strands of DNA). Single-strand breaks are
repaired by DNA ligase using the complementary strand of the double helix as a template,
with DNA ligase creating the final phosphodiester bond to fully repair the DNA.
DNA ligase is used in both DNA repair and DNA replication (see Mammalian ligases).
In addition, DNA ligase has extensive use in molecular biology laboratories for
recombinant DNA experiments (see Applications in molecular biology research). Purified
DNA ligase is used in gene cloning to join DNA molecules together to form recombinant
DNA.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
4. State the difference between 3 types of restriction endonuclease. (MAY’ 2010,
2012,2016)
ii. Comment on nomenclature of restriction enzyme. (MAY 2014, 2017)
iii. Mechanism of cutting by restriction enzyme (MAY 2017), (Dec' 2016)
iv. Explain type II restriction enzyme with suitable examples. (MAY 2015,2017),
(Dec' 2016)
i. Difference between 3 types of restriction endonuclease
Type I- Cleaves DNA upto l000b.p away
Type II-C leaves DNA at the target site away
TypeIll -Cleaves DNA upto 24-26b.p away
Also write in detail about properties of all enzymes
ii .Nomenclature of restriction enzymes 1.
Species name of the host organism is identified by the first two letters of the epithet to form
3letter
Abbreviation ex — Escherichia coli = Eco
2. Strain or type identification is written as subscript
eg — Eco k
3. When a particular host strain has severar different restriction and modification systems
these are identified by roman numerals ex — Hind I, Hind II, Hind Ill
iii. Mechanism of cutting by restriction enzyme
Blunt end – cut the sequences of DNA at the center of symmetry
Cohesive end- cut the sequences of DNA at the around of symmetry
Also in detail about the production of the blunt end and cohesive end molecules.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
5. What are some potential opportunities that can result from molecular
biotechnology? (DEC’2010)
Provides opportunity to accurately diagnose, prevent,cure wide range of diseases.
Significantly increases the crop yields by creating plants that are resisitant to
insect predation, fungal and viral diseases and environmental stresses, such as
short term drought and excessive heat and at the same time reduces application of
hazardous agri chemicals
Develop microorganisms that produces
antibiotics,chemicals,enzymes,aminoacids, and various food additives that
important food production and other industries
Develop livestock and other animals that have genetically enhanced attributes.
Facilitate removal of waste and other pollutants from the environment.
Biotechnology is a complement - not a substitute - for many areas of conventional
agricultural research. It offers a range of tools to improve our understanding and
management of genetic resources for food and agriculture. These tools are already
making a contribution to breeding and conservation programmes and to
facilitating the diagnosis, treatment and prevention of plant and animal diseases.
The application of biotechnology provides the researcher with new knowledge
and tools that make the job more efficient and effective. In this way,
biotechnology-based research programmes can be seen as a more precise
extension of conventional approaches (Dreher et al., 2000). At the same time,
genetic engineering can be seen as a dramatic departure from conventional
breeding because it gives scientists the power to move genetic material between
organisms that could not be bred through classical means.
Agricultural biotechnology is cross-sectoral and interdisciplinary. Most of the
molecular techniques and their applications are common across all sectors of food
and agriculture, but biotechnology cannot stand on its own. Genetic engineering
in crops, for example, cannot proceed without knowledge derived from genomics
and it is of little practical use in the absence of an effective plant-breeding
programme. Any single research objective requires mastery of a bundle of
technological elements. Biotechnology should be part of a comprehensive,
integrated agricultural research programme that takes advantage of work in other
sectoral, disciplinary and national programmes. This has broad implications for
developing countries and their development partners as they design and
implement national research policies, institutions and capacity-building
programmes .
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Agricultural biotechnology is international. Although most of the basic research
in molecular biology is taking place in developed countries, this research can be
beneficial for developing countries because it provides insight into the physiology
of all plants and animals. The findings of the human and the mice genome
projects provide direct benefits for farm animals, and vice versa, whereas studies
of maize and rice can provide parallels for applications in subsistence crops such
as sorghum and tef. However, specific work is needed on the breeds and species
of importance in developing countries. Developing countries are host to the
greatest array of agricultural biodiversity in the world, but little work has been
done on characterizing these plant and animal species at the molecular level to
assess their production potential and their ability to resist disease and
environmental stresses or to ensure their long-term conservation.
The application of new molecular biotechnologies and new breeding strategies to
the crops and livestock breeds of specific relevance to smallholder production
systems in developing countries will probably be constrained in the near future for
a number of reasons.
Refer molecular biotechnology by Bernard R. Glick, Jack J. Pasternak for
detailed.
.
Unit –II
Part -A
1. Difference between cDNA & Genomic library (May 2013), (DEC’ 2015) i. C-DNA – have only coding sequences.
ii. Genomic – have both coding & non-coding sequences.
2. What is meant by shotgun method? Where it is used?
Cutting the Genomic DNA using short restriction enzymes which are 4-6
bases long and is used to create genomic library.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
3. State the advantages of library Or Mention one application of cDNA & Genomic
library (May 2013, 2015), (Dec' 2016)
Store large amount of information for retrieval upon request.
Easy to start a new project at an advanced stage of cloning with the
retrieval of a readymade vector insert molecule.
4. Show the various types of vector with respect to size of the insert.
i. i) Plasmid vector – 5 kb ii) Phage – 10 kb
ii. iii) P1 phage – 100 kb iv) Cosmid – 45 kb
iii. v) BAC – 300 kb vi) YAC – 100 kb
5. What is meant by short gun method? Where it is used? (DEC’2013)
Cutting with short (4b.p.) recognition sequences so that a particular restriction site
is only occasionally cleaved and helps in the construction of a random genomic
library in which all fragment have same fragment ends thus helping in retrieval of
a fragments from the vector with the help of the same enzyme.
6. What is the difference between Southern, Northing and Western blotting.
i. Western blotting- detection of specific protein
ii. Southern blotting- detection of specific DNA sequences.
iii. Northern blotting- detection of specific mRNA
7. What are the different types of solid support used in blotting technique?
i. Western blotting - nitrocellulose membrane
ii. Southern blotting - nitrocellulose membrane
iii. Northern blotting - chemically reacted paper
8. Define blotting.
Blotting describes the immobilization of sample nucleic acids onto a solid support
(Nylon or nitrocellulose membrane).
9. Define autoradiography.
The localization and recording of a radiolabel within a solid specimen is known as
autoradiography.
10. Show the diagrammatic representation of hybridization.
i. 11. Define the term Hybridization and classify it.
a. Association of 2 complementary nucleic acid strands to form double stranded
b. molecules which can contain 2 DNA strands(DNA,DNA hybridization ), 2 RNA
strands (RNA, RNA hybridization), one DNA strand and one RNA strand.(DNA
RNA hybridization)
12. What is meant by Molecular probe? (DEC’2013), (Dec' 2016) a. Defined RNA or DNA fragment, radioactively or chemically labeled and is used
to
b. detect specific nuclei acid sequences by hybridization.
13. Mention the factors involved in Gene expression/Mention the various levels at which
a
gene can regulated (DEC’2010).
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Promoter’s strength, translational initiation sequences, codon size, secondary
structure
of mRNA , transcriptional termination, plasmid copy number ,plasmid stability
and
host cell physiology
14. Why is chemical method of DNA sequencing not popular? Discuss the reasons
(DEC’ 2010)
Chemical method of DNA can sequence only very short nucleotide bases of
approximately 500b.p and degradation of bases occurs. So it is not preferred.
15. How do ribosomal proteins control translation (MAY’ 2010)
A ribosomal protein is any of the proteins that, in conjunction with rRNA, make
up the ribosomal subunits involved in the cellular process of translation and have
control over translation. The proteins are denoted by S4, S7, S8, S15, S17, S20
bind independently to 16SrRNA.
16. What are the applications of PCR in the construction of cDNA and gDNA
libraries?(DEC’ 2015) Selectively amplify the target sequences directly from source of DNA using PCR
and cloned.
In PCR approach, screening step is built into 1st stage of the procedure so that
only selected fragments are actually cloned.
17. Write about S1 nuclease.(MAY 2014). S1 Nuclease is a single-strand-specific endonuclease that hydrolyzes single-
stranded RNA or DNA into 5 mononucleotides.
18. What are inverted repeats? (MAY’ 2015)
An inverted repeat (or IR) is a sequence of nucleotides followed downstream by
its reverse complement.The intervening sequence of nucleotides between the
initial sequence and the reverse complement can be any length including zero.
When the intervening length is zero, the composite sequence is a palindromic
sequence.
19. What is meant by chromosome jumping? (DEC’2013) Brings together DNA sequences that were originally located at considerable
distance apart in the genome and speeds up the process of long range
chromosome walking.
20. Draw a Bacmid and label its parts (MAY’ 2010)
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
PART-B
1. Describe the steps involved in the southern blotting using radioactive labeled
probe and non-radioactive probe to identify a DNA fragment. Discuss the major
differences between the southern and western blotting. (MAY’ 2013 )
A Southern blot is a method routinely used in molecular biology to check for
the presence of a DNA sequence in a DNA sample. Southern blotting combines
agarose gel electrophoresis for size separation of DNA.Steps involved in the
southern blotting
Restriction endonucleases are used to cut high-molecular-weight DNA strands
into smaller fragments. The DNA fragments are then electrophoresed on an
agarose gel to separate them by size. If some of the DNA fragments are larger
than 15 kb, then prior to blotting, the gel may be treated with an acid, such as
dilute HCl, which depurinates the DNA fragments, breaking the DNA into
smaller pieces, thus allowing more efficient transfer from the gel to membrane.
If alkaline transfer methods are used, the DNA gel is placed into an alkaline
solution (typically containing sodium hydroxide) to denature the double-
stranded DNA. The denaturation in an alkaline environment provides for
improved binding of the negatively charged DNA to a positively charged
membrane, separates it into single DNA strands for later hybridization to the
probe (see below), and destroys any residual RNA that may still be present in
the DNA.
A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of
(or below, depending on the direction of the transfer) the gel. Pressure is
applied evenly to the gel (either using suction, or by placing a stack of paper
towels and a weight on top of the membrane and gel), to ensure good and even
contact between gel and membrane. Buffer transfer by capillary action from a
region of high water potential to a region of low water potential (usually filter
paper and paper tissues) is then used to move the DNA from the gel on to the
membrane; ion exchange interactions bind the DNA to the membrane due to the
negative charge of the DNA and positive charge of the membrane.
The membrane is then baked, i.e., exposed to high temperature (60 to 100 °C)
(in the case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to
permanently and covalently crosslink the DNA to the membrane.
The membrane is then exposed to a hybridization probe—a single DNA
fragment with a specific sequence whose presence in the target DNA is to be
determined. The probe DNA is labelled so that it can be detected, usually by
incorporating radioactivity or tagging the molecule with a fluorescent or
chromogenic dye. In some cases, the hybridization probe may be made from
RNA, rather than DNA. To ensure the specificity of the binding of the probe to
the sample DNA, most common hybridization methods use salmon testes
(sperm) DNA for blocking of the membrane surface and target DNA, deionized
formamide, and detergents such as SDS to reduce non-specific binding of the
probe.
After hybridization, excess probe is washed from the membrane, and the pattern
of hybridization is visualized on X-ray film by autoradiography in the case of a
radioactive or fluorescent probe, or by development of color on the membrane
if a chromogenic detection method is used.
In case of non –radioactive probe HRP labelled or biotin labelled probes are
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
used which on treatment with suitable substrate gives a colored product(colored
band)
2.Write a note on the following methods used for screening the library southern –
western, northern –western, PCR method and functional complementation methods
of screening in detail. (MAY 2016), (DEC’ 2015) Southern –Western Screening - A closely related approach has been used for the
screening and isolation of clones expressing sequencespecific DNA-binding proteins.
The screening is carried out, without using an antibody, by incubating the membranes
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
with a radiolabelled doublestranded DNA oligonucleotide probe, containing the
recognition sequence for the target DNA-protein. This technique is called south-western
screening, because it combines the principles of Southern and western blots. It has been
particularly successful in the isolation of clones expressing cDNA sequences
corresponding to certain mammalian transcription factors
Northern –Western Screening - a similar technique as above and used to isolate
sequence specific RNA-binding proteins. In this case a single-stranded RNA probe is
being used. By analogy to the above,this is termed north-western screening and has been
successful in a number of cases.PCR method – amplification can be done using specific
primers to screen the gene of interest.functional complementation methods of screening
3.Elaborate in detail about screening of libraries using antisera. (MAY’ 2011,2016,DEC’
2015)
i.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
4.Elaborate in detail about screening of libraries using DNA probes. (MAY’ 2011, 2013,
2015,2016)
5.Write in detail about any two methods used for the synthesis of full length
double stranded cDNA.(MAY 2012),(DEC’ 2015)
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
6.Explain in detail how you would create a cDNA library and state its application. (MAY’ 2011 , 2012,
2013,2015, ,2017 ),(Dec' 2016)
steps involved in creating cDNA.
Isolation of mRNA.
Synthesis of cDNA using reverse transcriptase
Cloning using a vector molecule.
Purification of mRNA
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
7.Explain in detail how you would create a Genomic library and state its
application.(MAY’ 2011,2015,2016)
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Unit –III
Part -A
1. What are the requirements for a basic PCR reaction?
Template DNA, primers, dNTP, Taq polymerase, PCR buffers with mg+ ions
and sterile water.
2. Show the structure of a ddNTP.
3. What is the function of ddNTP.
a. They randomly incorporate at the positions of the corresponding
dNTP and such
b. addition of a ddNTP terminates polymerization because of the
absence of 3 hydroxyl
c. which prevents addition of the next nucleotide.
4. Can you analyze sequencing using automated system? Write its
advantages.
a. Yes.
b. Advantages: i) Speed ii) They help to minimize the ever-present
difficulty of clerical errors accumulating in sequencing
5. What the types of mutation?
Addition, Deletion and insertion.
6. What is meant by cassette mutagenesis?
In cassette mutagenesis a synthetic DNA fragment containing the desired mutant
sequence is used to replace the corresponding sequence in the wild type gene.
7. What are the steps involved in sanger di-deoxy method of sequencing.
a. Ability to synthesize faithfully a complementary copy of a single stranded DNA
template using a synthetic 5’end labeled oligodeoxynucleotide as primer.,
b. Polymerization using low concentration of one the 4ddNTPs and in higher
concentration of normal dNTPs,termination of growing point of the DNA chain
using 2’3’-dideoxy nucleotide triphosphate as substrate,
c. Separation of fragment using gel electrophoresis,
d. Analyzing the separated fragments using autoradiography.
8. What is meant by PCR and mention its use.
Polymerase chain reaction used for amplification of a specific DNA sequences by an
enormous factor.
9. Define primers.(MAY 2014) Short oligonucleotide bases used to initiate the DNA replication. They are 16-30 bases
long. Both forward and reverse primers are available
10. State the 3 steps involved in PCR.
i. i) Denaturation – 94ºc
ii. ii) Annealing – 55ºc
iii. iii) Extension – 72ºc
11. Classify site directed mutagenesis
Single primer method, strand selection method, transformation with oligonucleotides,
random mutagenesis, invitro strand selection and making unidirectional deletions
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
12. What is PCR assisted DNA sequencing? (DEC’ 2013) Sequencing by sangers dideoxy method is referred as PCR assisted DNA sequencing
13. Define site directed mutagenesis
A technique, which is used for introducing mutations at the desired place in a DNA
sequence by altering the sequences of primers
14. Why southern data necessary for designing an inverse PCR? (DEC’ 2010)
Inverse PCR is carriedout when the end sequence is unknown. In southern blotting the
hybridization probes helps in designing the primers.
15. What is meant by transposon tagging? (MAY’ 2010)
The term "transposon tagging" refers to a process in genetic engineering where
transposons (transposable elements) are amplified inside a biological cell by a tagging
techique. Transposon tagging has been used with several species to isolate genes.Even
without
16. What is nested PCR? When it is required (MAY’ 2011, 2012, 2013). Nested polymerase chain reaction is a modification of polymerase chain reaction
intended to reduce the contamination in products due to the amplification of
unexpected primer binding sites.
17. What are molecular beacons? (May 2013, 2014,2016,2017).
Molecular beacons are single-stranded oligonucleotide hybridization probes that form a
stem-and-loop structure. The loop contains a probe sequence that is complementary to
a target sequence, and the stem is formed by the annealing of complementary arm
sequences that are located on either side of the probe sequence. A fluorophore is
covalently linked to the end of one arm and a quencher is covalently linked to the end
of the other arm. Molecular beacons do not fluoresce when they are free in solution.
However, when they hybridize to a nucleic acid strand containing a target sequence
they undergo a conformational change that enables them to fluoresce brightly.
18. Comment on Assembly PCR.
Polymerase cycling assembly (or PCA, also known as Assembly PCR) is a method for
the assembly of large DNA oligonucleotides from shorter fragments. The process uses
the same technology as PCR, but takes advantage of DNA hybridization and annealing
as well as DNA polymerase to amplify a complete sequence of DNA in a precise order
based on the single stranded oligonucleotides used in the process. It thus allows for the
production of synthetic genes and even entire synthetic genomes.
19. Comment on applications of PCR.
20. What are the varients of PCR.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
21. What is meant by hot start PCR.
The hot start PCR is a modified form of Polymerase chain reaction (PCR) which
avoids a non-specific amplification of DNA by inactivating the taq polymerase at
lower temperature.
22. Mention the advantages and disadvantages of SYBR dye in PCR technique.
Advantages It can be used to monitor the amplification of any double-stranded DNA sequence.
No probe is required, which can reduce assay setup and running costs, assuming that
your PCR primers are well designed and your reaction is well characterized.
Disadvantage The primary disadvantage is that it may generate false positive signals; i.e., because the
SYBR® dye binds to any double-stranded DNA, it can also bind to nonspecific double-
stranded DNA sequences. Therefore, it is extremely important to have well-designed
primers that do not amplify non-target sequences, and that melt curve analysis be
performed.
23. Distinguish the Taq and Pfu polymerases. (DEC’2014).
24. Why Taqman assay is essential?
TaqMan probe-based assays are widely used in quantitative PCR in research and
medical laboratories:
Gene expression assays
Pharmacogenomics
Human Leukocyte Antigen (HLA) genotyping
Determine the viral load in clinical specimens (HIV, Hepatitis)
Bacterial Identification assays
DNA quantification
SNP genotyping
Verification of microarray results
25.What are the important characteristics of Taq polymerase.(MAY 2014),(Dec'
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Unit -IV
PART-A
1. Show the diagrammatic representation of nucleosome model.
2. Comment on Euchromatin
2016) Full-length Taq polymerase has 832 amino acids and a molecular weight of 94,000
daltons.
It has a specific activity of 292,000 units, with each unit adding 10 nmoles of de
oxyribonucleotide triphosphate (dNTPs) into a product in 30 minutes. Taq was the
first polymerase found to retain its activity after exposure to high temperature. Most
specifically, Taq polymerase has an activity half-life of 45 to 50 minutes at 95
degrees C and of 9 minutes at 97.5 degrees C.
26. What are the methods to confirm the PCR product is correct or not.(DEC’ 2015) By running the PCR product along with DNA ladder using agarose gel
electrophoresis and
by using sequencing techniques.
27. What are the advantages of real-time PCR than end-point PCR.(DEC’ 2015) The measurement is made after each amplification cycle, and this is the reason why
this
method is called real time PCR (that is, immediate or simultaneous PCR) than end-
point
PCR.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
3. What are telomerases?
Replication of the telomere region occur in the presence of an enzyme called telomerases, it
uses
3’OH of a GC rich telomeric strand as a primer for the synthesis of tandem repeats.
4. Define Pyrosequencing. (JAN’ 2014)
Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in
DNA)
based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it
relies on the detection of pyrophosphate release on nucleotide incorporation, rather than
chain
termination with dideoxynucleotides.
5. Listout the advantages Pyrosequencing.
Advantages:
Accurate
Parallel processing
Easily automated
Eliminates the need for labeled primers and nucleotides
No need for gel electrophoresis
6. Comment on shot gun method of sequencing.
Used to sequence whole genomes
Steps:
DNA is broken up randomly into smaller fragments
Dideoxy method produces reads
Look for overlap of reads
7. Listout the advantages and disadvantages shotgun method of sequencing.
Advantages:
Fast sequencing at a high volume
Cheap compared to other methods
Much higher coverage protection
Disadvantages:
Repetitive sequences can disrupt computer program into thinking that unrelated
sequences
are in fact connected.
More prone to error and missing sequences
8. List out the emerging sequencing methods. (JAN 2015)
Sequencing by Hybridization (SBH).
Mass Spectrophotometric Sequences.
Direct Visualization of Single DNA Molecules by Atomic force Microscopy (AFM )
Single Molecule Sequencing Techniques
Single nucleotide Cutting
Nanopore sequencing
Readout of Cellular Gene Expression.
9. Comment on importance of sequencing. (JAN’ 2014) DNA sequencing helps us understand the essential genetic make-up of organisms.
10. Define de novo sequencing
The term "de novo sequencing" specifically refers to methods used to determine the
sequence of DNA with no previously known sequence. De novo translates from Latin as
"from the beginning".
11. Comment on Sequencing by hybridization
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A
single
pool of DNA whose sequence is to be determined is fluorescently labeled and
hybridized
to an array containing known sequences. Strong hybridization signals from a given spot
on the array identifies its sequence in the DNA being sequenced.
12. Mention the application of sequencing.
DNA sequencing may be used to determine the sequence of individual genes, larger genetic
regions (i.e. clusters of genes or operons), full chromosomes or entire genomes. Sequencing
provides the order of individual nucleotides in present in molecules of DNA or RNA
isolated
from animals, plants, bacteria, archaea, or virtually any other source of genetic information.
This
information is useful to various fields of biology and other sciences, medicine, forensics, and
other areas of study.
13. List out the Emerging Sequence Methods.
Sequencing by Hybridization (SBH).
Mass Spectrophotometric Sequences.
Direct Visualization of Single DNA Molecules by Atomic force Microscopy (AFM )
Single Molecule Sequencing Techniques
Single nucleotide Cutting
Nanopore sequencing
Readout of Cellular Gene Expression
14. State the Advantages & Disadvantages of Hierarchical Sequencing
Hierarchical Sequencing
– ADV. Easy assembly
– DIS. Build library & physical map;
redundant sequencing
Whole Genome Shotgun (WGS)
– ADV. No mapping, no redundant sequencing
– DIS. Difficult to assemble and resolve repeats
15. List out the steps to assemble a Genome.
16. Comment on top down and bottom up approach of sequencing.
• Top-down approach - Clone large genomic DNA fragments into special vector,
e.g. BAC (bacterial artificial chromosome)
- Create an ordered array of BAC clones
- Carry out full-length BAC clone sequencing
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
- Assemble the BAC insert sequences
- Identify the next BAC for full length sequencing
(Hybridization method or searching BAC end sequence library)
• Bottom-up approach - Whole genome shotgun sequencing
17. Mention the importance of DNA sequencing.
Knowledge of DNA sequences has become indispensable for basic biological research,
and in numerous applied fields such as
diagnostic,
biotechnology,
forensic biology, and
biological systematics.
The rapid speed of sequencing attained with modern DNA sequencing technology has
been instrumental in the sequencing of complete DNA sequences, or genomes of
numerous types and species of life, including the human genome and other complete
DNA sequences of many animal, plant, and microbial species.
18. List out the application of sequencing.
DNA sequencing may be used to determine the sequence of individual genes,
• larger genetic regions (i.e. clusters of genes or operons),
• full chromosomes or entire genomes.
Depending on the methods used, sequencing may provide the order of nucleotides in
DNA or RNA isolated from cells of animals, plants, bacteria, archaea, or virtually any
other source of genetic information.
The resulting sequences may be used by researchers in molecular biology or genetics to
further scientific progress or may be used by medical personnel to make treatment
decisions or aid in genetic counseling.
19. Listout the applications of about next generation sequencing.
20. Differentiate between Genetic mapping and Physical mapping (Dec 2014)
There are two distinctive types of "Maps" used in the field of genome mapping: genetic maps
and
physical maps. While both maps are a collection of genetic markers and gene loci, genetic
maps'
distances are based on the genetic linkage information, while physical maps use actual
physical
distances usually measured in number of base pairs. While the physical map could be a more
"accurate" representation of the genome, genetic maps often offer insights into the nature of
different regions of the chromosome, e.g. the genetic distance to physical distance ratio
varies
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
greatly at different genomic regions which reflects different recombination rates, and such
rate is
often indicative of euchromatic (usually gene-rich) vs heterochromatic (usually gene poor)
regions of the genome.
21. Define STS tag site. (May 2017) A sequence-tagged site (or STS) is a short (200 to 500 base pair) DNA sequence that has a
single
occurrence in the genome and whose location and base sequence are known.
22. List out the application of restriction enzyme Finger Printing.
Conceptual
1. That the uniqueness of genetic profiles can be exploited to distinguish individual
organisms.
2. That restriction endonucleases are enzymes that cleave DNA at specifically recognized
target
sequences.
3. That agarose gel electrophoresis is the use of a solid matrix and electrical current to
separate
DNA molecules by size.
Practical
To gain experience performing enzyme reactions and agarose gel electrophoresis.
To become familiar with the use of common laboratory equipment and to conduct
enzyme reactions and agarose gel electrophoresis, which will include pipettors, balances,
water baths, electrophoresis gel boxes, ultraviolet transilluminator.
To learn the proper handling procedures for enzymes and DNA and biohazardous wastes.
23. Listout the application of Genetic fingerprinting.
DNA analysis can be used for catching criminals, establishing parentage, finding how closely
organisms are related and many other applications.
24. Define Mapping and mention its application.
Mapping is the identification of genes and their positions in the chromosome. Modern
biochemical techniques are used to identify genes and their positions in the chromosome.
Special
staining methods reveal bands in the chromosomes. These do not necessarily represent genes
but
help to identify the position of genes
25. Comment on hybridization mapping.
Hybridization mapping makes use of the fact one can test a clone for the presence of small
known
genomic sequences (e.g. using a hybridization experiment or PCR).It is useful to distinguish
between unique probes such as STS (sequence tagged sites) and non-unique probes, as they
give
rise to different algorithmic problems. We will concentrate on unique probes.Given a set of
unique
probes, two protocols are commonly used, STS content mapping and radiation hybrid
mapping.
The goal is here to determine the order of overlapping clones. The hybridization signature of
short
(possibly unique) sequences is determined. The goal is to determine a possibly minimal tiling
path
so that only few clones need to be sequenced.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
PART- B
1. Explain in detail about shotgun method of sequencing. (JAN’2014), (MAY 2014,
2016, 2017).
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Advantages & Disadvantages of Hierarchical Sequencing
Hierarchical Sequencing
ADV. Easy assembly
DIS. Build library & physical map;
redundant sequencing
Whole Genome Shotgun (WGS)
ADV. No mapping, no redundant sequencing
DIS. Difficult to assemble and resolve repeats
The Walking method – motivation
1. Sequence the genome clone-by-clone without a physical map
2. The only costs involved are:
Library of end-sequenced clones (cheap)
Sequencing
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
2. Explain in detail about top down and bottom method of genome sequencing.(JAN’,
2014, 2015),(MAY 2014).
Genome Sequencing Strategies
Top-down approach Clone large genomic DNA fragments into special vector,
e.g. BAC (bacterial artificial chromosome)
- Create an ordered array of BAC clones
- Carry out full-length BAC clone sequencing
- Assemble the BAC insert sequences
- Identify the next BAC for full length sequencing
(Hybridization method or searching BAC end sequence library)
Bottom-up approach - Whole genome shotgun sequencing
Top-down genome sequencing method
Method I. Systematic sequencing of ordered clones
Construct shotgun genomic library in YAC (yeast artificial chromosome) or BAC vector
Use the YAC or BAC clone DNAs to construct smaller insert shotgun cosmid DNA
library (~45 kb inserts)
Multiple Complete Digest (MCD) mapping of cosmid DNAs ordered cosmid clone
library. Choose the minimal overlap set of cosmid DNA to construct shotgun libraries in
M13 or plasmid vector DNA sequencing Assembly
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
3. Explain in detail about next generation sequencing using suitable diagrams.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
4. Write short notes on
- Ordering the genome sequence
- Genetic maps and Physical maps
Ordering the genome sequence
Genetic maps and Physical maps
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
A genome map may be defined as a detailed schematic description of the structural and
functional organisation of all the chromosomes in the genome of an organism. The
genome, in turn, may be looked at in two different ways. To a cytogeneticist, it represents
the haploid set of chromosomes of a diploid organism.
Therefore, a polyploid organism will have more than two genomes, which may be
identical (autopolyploids) or distinct (allopolyploids).
But to a molecular biological genome consists of the total genetic information present in
the organism. At present, we have mainly three types of maps:
(i) genetic or, linkage maps,
(ii) cytogenetic maps, and
(iii) physical maps.
Genetic linkage map is a term which describes the tendency of certain loci or alleles to be
inherited together. Genetic loci on the same chromosome are physically close to one
another and tend to stay together during meiosis, and are thus genetically linked.
By working out the number of recombinants it is possible to obtain a measure for the
distance between the genes. This distance is called a genetic map unit (m.u.), or a
centimorgan and is defined as the distance between genes for which one product of
meiosis in 100 is recombinant. A recombinant frequency (RF) of 1 % is equivalent to 1
m.u.
Various physical mapping techniques are used like
STS: These are short sequences of DNA occur only once in the human genome, it is
exactly 200 to 300 basepair long, each STS is unique and helpful in physical map
construction.
Restriction map and cDNA map - different enzymes are used to create restriction maps
and complementary DNAs prepared and cloned into hosts can also be used for map
construction, ESTs, VNTRs, etc
In physical mapping, the DNA is cut by a restriction enzyme. Once cut, the DNA
fragments are separated by electrophoresis. The resulting pattern of DNA migration (i.e.,
its genetic fingerprint) is used to identify what stretch of DNA is in the clone. By
analysing the fingerprints, contigs are assembled by automated (FPC) or manual means
(Pathfinders) into overlapping DNA stretches. Now a good choice of clones can be made
to efficiently sequence the clones to determine the DNA sequence of the organism under
study (seed picking).
Macrorestriction is a type of physical mapping wherein the high molecular weight DNA
is digested with a restriction enzyme having a low number of restriction sites.
There are alternative ways to determine how DNA in a group of clones overlap without
completely sequencing the clones. Once the map is determined, the clones can be used as
a resource to efficiently contain large stretches of the genome. This type of mapping is
more accurate than genetic maps.
Genes can be mapped prior to the complete sequencing by independent approaches
like in situ hybridization.
Ex. Restriction map, EST map,etc.
5. Explain in detail about Restriction Enzyme Finger Printing.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
6. Write short notes on
- Radiation Hybrid Maps
- Optical mapping
Radiation Hybrid Maps
Radiation hybrid mapping is a genetic technique that was originally developed for
constructing long-range maps of mammalian chromosomes . It is based on a statistical
method to determine not only the distances between deoxyribonucleic acid (DNA)
markers but also their order on the chromosomes. DNA markers are short, repetitive
DNA sequences, most often located in noncoding regions of the genome , that have
proven extremely valuable for localizing human disease genes in the genome.
Theory and Application
In radiation hybrid mapping, human chromosomes are separated from one another and
broken into several fragments using high doses of X rays. Similar to the underlying
principle of mapping genes by linkage analysis based on recombination events, the
farther apart two DNA markers are on a chromosome, the more likely a given dose of X
rays will break the chromosome between them and thus place the two markers on two
different chromosomal fragments. The order of markers on a chromosome can be
determined by estimating the frequency of breakage that, in turn, depends on the distance
between the markers. This technique has been used to construct whole-genome radiation
hybrid maps.
Technique
A rodent-human somatic cell hybrid ("artificial" cells with both rodent and human
genetic material), which contains a single copy of the human chromosome
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Radiation hybrid mapping process.
of interest, is X-irradiated. This breaks the chromosome into several pieces, which are
subsequently integrated into the rodent chromosomes. In addition, the dosage of radiation
is sufficient to kill the somatic cell hybrid or donor cells, which are then rescued by
fusing them with nonirradiated rodent recipient cells. The latter, however, lack an
important enzyme and are also killed when grown in a specific medium. Therefore, the
only cells that can survive the procedure are donor-recipient hybrids that have acquired a
rodent gene for the essential enzyme from the irradiated rodent-human cell line
Optical mapping
Optical mapping is a technique for constructing ordered, genome-wide, high-resolution
restriction maps from single, stained molecules of DNA, called "optical maps". By
mapping the location of restriction enzyme sites along the unknown DNA of an
organism, the spectrum of resulting DNA fragments collectively serve as a unique
"fingerprint" or "barcode" for that sequence. Originally developed by Dr. David C.
Schwartz and his lab at NYU in the 1990s this method has since been integral to the
assembly process of many large-scale sequencing projects for both microbial and
eukaryotic genomes.
The modern optical mapping platform works as follows
Genomic DNA is obtained from lysed cells, and randomly sheared to produce a "library"
of large genomic molecules for optical mapping.
A single molecule of DNA is stretched (or elongated) and held in place on a slide under a
fluorescent microscope due to charge interactions.
DNA molecule is digested by added restriction enzymes, which cleave at specific
digestion sites. The resulting molecule fragment remain attached to the surface. The
fragment ends at the cleavage site are drawn back (due to elasticity of linearized DNA),
leaving gaps which are identifiable under the microscope as gaps.
DNA fragments stained with intercalating dye are visualized by fluorescence microscopy
and are sized by measuring the integrated fluorescence intensity. This produces an optical
map of single molecules.
Individual optical maps are combined to produce a consensus, genomic optical map.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
b.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
UNIT 5
PART A
1. Define the term genome and Genomics.(NOV’13)(MAY’14)
Genome means haploid DNA content of an organism.
The complete, genetic complement of a virus, cell or organism.is called as genomics.
2. Define Structural genomics.
It is also called as the classical genomics. In this approach, genetic mapping, physical
mapping and the complete sequencing is done.
3. Comment on Human Genome project. It is a worldwide research effort initiated by the department of Energy and National Institute
of Health in 1987. Primary goal is to produce chromosome map and sequence each
chromosome of a man.
4 . What is functional genomics? (NOV’13), (May 2016)
The determination of specific functions of different expressed sequence is called functional
genomics to find the function of new genes.
5. What are the goals of functional Genomics?
Understand the relationships between the organism’s genome and the phenotype.
Understand the dynamic properties of an organism.
Study the mutation, study the pattern of gene expression, etc.
6. Explain Y2H and its role in functional genomics?
Two-hybrid screening (also known as yeast two-hybrid system or Y2H) is a molecular biology
technique used to discover protein-protein interactions[1] and protein-DNA interactions by
testing
for physical interactions (such as binding) between two proteins or a single protein and a DNA
molecule, respectively.
7. What are DNA chips/micro arrays?
Microarrays is a high-density miniaturized arrays of molecular samples, facilitating the
screening of
genomic DNA or cDNA samples, facilitating the screening of genomic DNA or cDNA samples
for
the presence of one in 1,00,000 or more DNA sequences.
8. What do you mean by antibody microarray?
An antibody microarray is a specific form of protein microarrays, a collection of capture
antibodies
are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose
of
detecting antigens. Antibody microarray is often used for detecting protein expressions from
cell
lysates in general research and special biomarkers from serum or urine for diagnostic
applications
9. What is meant by miRNA microarray?
MicroRNAs (miRNAs) are short ribonucleic acid (RNA) molecules, on average only 22
nucleotides
long and are found in alleukaryotic cells. miRNAs are post-transcriptional regulators that bind
to
complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
translational repression and gene silencing. The arrays based on miRNA is called as miRNA
array.
10. Mention the various methods adopted for the fabrication of microarray?
Inkjet printing, photolithography and electrochemical methods.
11. Write short notes on SAGE and its role?
Serial analysis of gene expression (SAGE) is a technique used by molecular biologists to
produce a
snapshot of the messenger RNA population in a sample of interest in the form of small tags
that
correspond to fragments of those transcripts.
12. Comment on TOGA? (Nov’13), (May 2016,2017) A completely automated technology for the simultaneous analysis of the expression of nearly
all
genes. Basically, it selects a four-base recognition endonuclease site and an adjacent four
nucleotide parsing sequence (a syntactical determinant, e.g., for MspI CCGGN1N2N3N4)
and
their distance from the 3′-end of an mRNA (from the polyA tail).
13. Define DNA microarray.
A DNA microarray (also commonly known as DNA chip or biochip) is a collection of
microscopic DNA spots attached to a solid surface.
14. List out the application of DNA microarray. (May 2014) Gene expression profiling
Comparative genomic hybridization
Chromatin immunoprecipitation on Chip
SNP detection
Alternative splicing detection
Fusion genes microarray
Tiling array
15. Define Subtractive hybridization
Subtractive hybridization is a technology that allows for PCR-based amplification of only
cDNA
fragments that differ between a control (driver) and experimental transcriptome.
16. What is meant by DIGE.
Difference gel electrophoresis (DIGE) is a form of gel electrophoresis where up to three
different
protein samples can be labeled with size-matched, charge-matched spectrally resolvable
fluorescent dyes (for example Cy3, Cy5, Cy2) prior to two-dimensional electrophoresis.
17. Define Comparative genomics.
Comparative genomics is a field of biological research in which the genomic features of
different
organisms are compared. The genomic features may include the DNA sequence, genes, gene
order, regulatory sequences, and other genomic structural landmarks.
18. Define Proteogenomics
Proteogenomics is an area of research at the interface of proteomics and genomics.
19. What does proteogenomics offer?
Accurate prediction of Translation Start Site.
Accurate prediction of programmed frameshifts.
Accurate prediction of post translational modification.
A confirmation if a (pseudo)gene is actually translated.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
20. What does proteogenomics struggle with?
For a novel protein, mapping the peptides from the Mass Spectrometry experiments
to the exomes/genomes (similar problem as RNA-Seq)
Currently they try to collect exomes (regions that is assumed to be exons) and
translate them in 6 different frames (3 in each DNA strand).
They also build a exon splice graph which models different splicing alternatives of a
single gene
21.Describe the significance of fluorescent hybridization. (May 2017) o Helps in diagnosis of a pathogenic molecule.
o Helps in forensic studies.
o Helps in blotting technique.
22. Mention the significance of 2D gel electrophoresis. (May 2017) Used separate 2 different molecule having same molecular weight in the basis of isoelectric
point
and molecular weight.
23. Although all human cells do have same genes, they are not identical in their expression.
Why?(May 2017)
Because of the SNP's existing for every thousand base pairs and environmental condition for
the
expression of the genes.
24. Outline the principle of microarrays. (May 2017)
PART B
1. What is functional genomics and explain the various techniques used for the study of
functional genomics.
It is the study of all specific genes and their expression in time and space in an organism.
Goals:
Understand the relationships between the organism’s genome and the phenotype.
Understand the dynamic properties of an organism.
Study the mutation.
Study the pattern of gene expression, etc.
Techniques:
Functional genomics includes function-related aspects of the genome itself such as mutation
and polymorphism (such as SNP) analysis, as well as measurement of molecular activities.
The latter comprise a number of "-omics" such as transcriptomics (gene expression),
proteomics (protein expression), phosphoproteomics (a subset of proteomics) and
metabolomics. Functional genomics uses mostly multiplex techniques to measure the
abundance of many or all gene products such as mRNAs or proteins within a biological
sample. Together these measurement modalities endeavor to quantitate the various biological
processes and improve our understanding of gene and protein functions and interactions.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
It can be studied at 1).DNA level
2) RNA level – SAGE, Microarray, etc.
3). Protein level – Y2H, Protein array, etc.
2. Explain SAGE in detail. (May 2011, 2016).
Serial analysis of gene expression (SAGE) is a technique used by molecular biologists to
produce a snapshot of the messenger RNA population in a sample of interest in the form of
small tags that correspond to fragments of those transcripts. The original technique was
developed by Dr. Victor Velculescu at the Oncology Center of Johns Hopkins University and
published in 1995[1]. Several variants have been developed since, most notably a more robust
version, LongSAGE[2], RL-SAGE[3] and the most recent SuperSAGE[4]. Many of these have
improved the technique with the capture of longer tags, enabling more confident identification
of a source gene.
Overview
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Briefly, SAGE experiments proceed as follows:
1. Isolate the mRNA of an input sample (e.g. a tumour).
2. Extract a small chunk of sequence from a defined position of each mRNA molecule.
3. Link these small pieces of sequence together to form a long chain (or concatemer).
4. Clone these chains into a vector which can be taken up by bacteria.
5. Sequence these chains using modern high-throughput DNA sequencers.
6. Process this data with a computer to count the small sequence tags.
A more in-depth, technical explanation of the technique is available here
Recent sage applications
Analysis of yeast transcriptome
Gene Expression Profiles in Normal and Cancer Cell
Insights into p53-mediated apoptosis
Identification and classification of p53-regulated genes
Analysis of human transcriptomes
Serial microanalysis of renal transcriptomes
Genes Expressed in Human Tumor Endothelium
Analysis of colorectal metastases (PRL-3)
Characterization of gene expression in colorectal adenomas and cancer
Using the transcriptome to analyze the genome (Long SAGE)
3. Explain in detail about DNA microarray and its applications.
A DNA microarray is a multiplex technology used in molecular biology. It consists of an
arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features,
each containing picomoles (10−12 moles) of a specific DNA sequence, known
as probes (or reporters). These can be a short section of agene or other DNA element that are
used to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions.
Probe-target hybridization is usually detected and quantified by detection of fluorophore-,
silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid
sequences in the target. Since an array can contain tens of thousands of probes, a microarray
experiment can accomplish many genetic tests in parallel. Therefore arrays have dramatically
accelerated many types of investigation.
In standard microarrays, the probes are attached via surface engineering to a solid surface by
a covalent bond to a chemical matrix (via epoxy-silane, amino-
silane, lysine, polyacrylamide or others). The solid surface can be glass or a silicon chip, in
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
which case they are colloquially known as an Affy chip when anAffymetrix chip is used.
Other microarray platforms, such asIllumina, use microscopic beads, instead of the large
solid support. DNA arrays are different from other types of microarray only in that they
either measure DNA or use DNA as part of its detection system.
DNA microarrays can be used to measure changes in expressionlevels, to detect single
nucleotide polymorphisms (SNPs), or to genotype or resequence mutant genomes (see uses
and typessection). Microarrays also differ in fabrication, workings, accuracy, efficiency, and
cost (see fabrication section). Additional factors for microarray experiments are the
experimental design and the methods of analyzing the data (see Bioinformaticssection).
Several Types of Arrays
Spotted DNA arrays
Developed by Pat Brown‟s lab at Stanford
PCR products of full-length genes (>100nt)
Affymetrix gene chips
Photolithography technology from computer industry allows building many 25-
mers
Ink-jet microarrays from Agilent
25-60-mers “printed directly on glass slides
Flexible, rapid, but expensive
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
DNA microarrays can be manufactured by:
Photolitography (Affymetrix, Febit, Nimblegen)
Inkjet (Agilent, Canon)
Robot spotting (many providers)
Array Fabrication Spotting
Use PCR to amplify DNA
Robotic "pen" deposits DNA at defined coordinates
approximately 1-10 ng per spot
Experimentation with oligos (40, 70 bp)
Array Fabrication Photolithography
Light activated synthesis
synthesize oligonucleotides on glass slides
107
copies
per oligo in 24 x 24 um square
Use 20 pairs of different 25-mers per gene
Perfect match and mismatch
Microarray: Used for
– Genome-wide studies and genotyping
– Evaluating microRNA levels
– Gene expression profiling
– Comparative genomic hybridization
– Chromatin immunoprecipitation on Chip
– SNP detection
– Alternative splicing detection
– Fusion genes microarray
– Tiling array
4. Define the hydrization and explain in detail about Subtractive hybridization (May
2016,2017).
Subtractive cloning is a powerful technique for isolating genes expressed or present in one cell
population but not in another. This method and a related one termed positive selection have their
origins in nucleic acid reassociation techniques. We discuss the history of subtractive techniques,
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
and fundamental information about the nucleic acid composition of cells that came out of
reassociation analyses. We then explore current techniques for subtractive cloning and positive
selection, discussing the merits of each. These techniques include cDNA library–based
techniques and PCR-based techniques. Finally, we briefly discuss the future of subtractive
cloning and new approaches that may augment or supersede current methods.
Subtractive cloning uses a process called driver excess hybridization. Nucleic acid from
which one wants to isolate differentially expressed sequences (the tracer) is hybridized to
complementary nucleic acid that is believed to lack sequences of interest (the driver). Driver
nucleic acid is present at much higher concentration (at least 10-fold) than is tracer, and it
dictates the speed of the reannealing reaction. The driver and tracer nucleic acid populations
are allowed to hybridize, and only sequences common to the two populations can form
hybrids. After hybridization, driver-tracer hybrids and unhybridized driver are removed. This
is the subtraction step. The tracer that remains behind is enriched for sequences specific to
the tracer tissue source [often called the plus (C) source] and depleted for sequences common
to tracer and driver [often called the minus (−) source]. Usually, the process must be
performed reiteratively in order to remove all the sequences common to both the driver and
the tracer. After subtraction, remaining nucleic acid can be used to prepare a library enriched
in tracer-specific clones or to make a probe that can be used to screen a library for tracer-
specific clones.
5. Write short notes on
i. DIGE
ii. TOGA(MAY 2017)
i. DIGE
Allows the separation of treated (or diseased) and untreated (or control) samples in a
single physical gel.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Quick comparison in the differences of the protein profiles of each sample by overlaying
the unwrapped maps of treated and untreated samples. It is possible to see which proteins
are shared by both, which are present in one sample but not in the other.
In a DIGE system, proteins are pre-labelled with fluorescent CyDyes™ such as Cy3, and
Cy5 prior to electrophoretic separations. Labelled samples are then mixed before
isoelectric focusing, and resolved on the same 2D gel.
Key benefits:
More confidence- reflects true biological outcomes and is not due to the technical
variation
Less gels- saves time by reducing the large number of replicates that are used in the
conventional, single stain 2D gel method
High accuracy- no false negative and no false positive
Quantitative data In a new DIGE system, proteins are pre-labelled with fluorescent CyDyes™ such as Cy2,
Cy3, and Cy5 prior to electrophoretic separations. Labelled samples are then mixed
before isoelectric focusing, and resolved on the same 2D gel.
Cy2 dye is used to label an internal standard, which consists of a pooled sample
comprising of equal amounts of each of the samples to be compared. This allows both
inter and intra gel matching, and is used in the standardization of spot volumes in
different gels. Spot volumes are expressed as a ratio to the internal standard.
Images of each dye are acquired with various lasers using a variable mode imager and
images are analyzed with differential image analysis software.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
6. Explain in detail about Yeast Two hybrid System.
Y2H:
Two-hybrid screening (also known as yeast two-hybrid system or Y2H) is a molecular biology
technique used to discover protein-protein interactions[1] and protein-DNA interactions[2][3] by
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
testing for physical interactions (such as binding) between two proteins or a single protein and a
DNA molecule, respectively.
The premise behind the test is the activation of downstream reporter gene(s) by the binding of a
transcription factor onto an upstream activating sequence (UAS). For two-hybrid screening, the
transcription factor is split into two separate fragments, called the binding domain (BD) and
activating domain (AD). The BD is the domain responsible for binding to the UAS and the AD is
the domain responsible for the activation of transcription.
The two-hybrid system is a useful way to detect proteins that interact with a protein you are
studying. In general, it is used primarily for initial identification of interacting proteins, not for
detailed characterization of the interaction.
This system is based on the modular organization of many transcription factors (see figure).
Many such proteins have two or more discrete structural and functional units, or domains. The
first protein to be used for this was the yeast protein GAL4; many later studies use the DNA-
binding domain of the E. coli protein LexA. GAL4 has a DNA-binding domain (or DBD) and an
activation domain (or AD). The structure of GAL4 complexed to its specific site (PDB file) is
only of the first 65 amino acids, and comprises a minimal DBD; usually residues 1-100 or so are
used.
When GAL4 binds to its cognate binding site, the activation domain is brought close to the
promoter, allowing the activation domain to interact with the transcription machinery and
resulting in activation of transcription. Typically a reporter gene, often lacZ, is used. Hence,
there are standard reporter constructs, with variable numbers of binding sites and a reporter gene.
Now consider how these elements can be used to detect protein-protein interactions. Two types
of hybrids are made:
DBD Hybrid: This hybrid contains the DBD fused to a protein of interest (often termed the
"bait"). This fusion protein can bind to the DNA, but cannot activate transcription because the
bait does not contain an activation function (if it does, this procedure will not work).
AD Hybrid: This hybrid contains the AD fused to another protein (often termed the "prey").
Usually, a recombinant DNA "library" is prepared in which genes for many different proteins are
fused to the AD. Then both hybrid proteins are expressed in the same cell. Those expressing the
reporter gene are identified and purified for further characterization.
Typically, libraries will contain large numbers of different clones (>106 different ones); a few of
them will be able to interact with the bait. These few can then be recognized by their ability to
turn on the reporter gene.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19