DEPARTMENT OF BIOTECHNOLOGY · 2019-11-22 · BT 6603 Genetic Engg and Genomics (VI sem) Department...

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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

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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 .

    http://www.greenfacts.org/glossary/abc/biotechnology.htmhttp://www.greenfacts.org/glossary/ghi/genetic-engineering.htmhttp://www.greenfacts.org/glossary/ghi/genetic-material.htmhttp://www.greenfacts.org/glossary/abc/biotechnology.htmhttp://www.greenfacts.org/glossary/ghi/genetic-engineering.htmhttp://www.greenfacts.org/glossary/ghi/genomics.htm

  • 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.

    http://www.greenfacts.org/glossary/abc/biotechnology.htmhttp://www.greenfacts.org/glossary/pqrs/physiology.htmhttp://www.greenfacts.org/glossary/ghi/genome.htmhttp://www.greenfacts.org/glossary/pqrs/species.htmhttp://www.greenfacts.org/glossary/abc/biotechnology.htm

  • 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

    http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Agarose_gel_electrophoresishttp://en.wikipedia.org/wiki/Electrophoresishttp://en.wikipedia.org/wiki/Endonucleasehttp://en.wikipedia.org/wiki/HClhttp://en.wikipedia.org/wiki/Depurinationhttp://en.wikipedia.org/wiki/Sodium_hydroxidehttp://en.wikipedia.org/wiki/Hybridisation_(molecular_biology)http://en.wikipedia.org/wiki/Nitrocellulosehttp://en.wikipedia.org/wiki/Nylonhttp://en.wikipedia.org/wiki/Artificial_membranehttp://en.wikipedia.org/wiki/Capillary_actionhttp://en.wikipedia.org/wiki/Water_potentialhttp://en.wikipedia.org/wiki/Ion_exchangehttp://en.wikipedia.org/wiki/Ultraviolet_radiationhttp://en.wikipedia.org/wiki/Covalenthttp://en.wikipedia.org/wiki/Hybridization_probehttp://en.wikipedia.org/wiki/Radioactivityhttp://en.wikipedia.org/wiki/Fluorescencehttp://en.wikipedia.org/wiki/Formamidehttp://en.wikipedia.org/wiki/Sodium_dodecyl_sulfatehttp://en.wikipedia.org/wiki/X-rayhttp://en.wikipedia.org/wiki/Autoradiography

  • 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.

    http://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Electrophoresishttp://en.wikipedia.org/wiki/Genetic_fingerprinthttp://en.wikipedia.org/wiki/Clone_(genetics)http://en.wikipedia.org/wiki/Contighttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/In_situ_hybridization

  • 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

    http://www.biologyreference.com/Fo-Gr/Genome.html

  • 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

    http://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/Biologisthttp://en.wikipedia.org/wiki/Messenger_RNAhttp://en.wikipedia.org/wiki/Victor_Velculescuhttp://en.wikipedia.org/wiki/Johns_Hopkins_Universityhttp://en.wikipedia.org/wiki/Serial_analysis_of_gene_expression#cite_note-SAGE-0http://en.wikipedia.org/wiki/Serial_analysis_of_gene_expression#cite_note-Saha-1http://en.wikipedia.org/w/index.php?title=RL-SAGE&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=RL-SAGE&action=edit&redlink=1http://en.wikipedia.org/wiki/SuperSAGEhttp://en.wikipedia.org/wiki/SuperSAGE

  • 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

    http://en.wikipedia.org/wiki/MRNAhttp://en.wikipedia.org/wiki/Tumourhttp://en.wikipedia.org/wiki/MRNAhttp://en.wikipedia.org/wiki/Vector_(molecular_biology)http://www.sagenet.org/http://en.wikipedia.org/wiki/Multiplex_(assay)http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Oligonucleotidehttp://en.wikipedia.org/wiki/Picohttp://en.wikipedia.org/wiki/Picohttp://en.wikipedia.org/wiki/Hybridization_probehttp://en.wikipedia.org/wiki/Genehttp://en.wikipedia.org/wiki/Nucleic_acid_hybridizationhttp://en.wikipedia.org/wiki/CDNAhttp://en.wikipedia.org/wiki/Fluorophorehttp://en.wikipedia.org/wiki/Genetic_testhttp://en.wikipedia.org/wiki/Surface_engineeringhttp://en.wikipedia.org/wiki/Covalent_bondhttp://en.wikipedia.org/wiki/Epoxyhttp://en.wikipedia.org/wiki/Aminehttp://en.wikipedia.org/wiki/Lysinehttp://en.wikipedia.org/wiki/Polyacrylamidehttp://en.wikipedia.org/wiki/Glass

  • 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

    http://en.wikipedia.org/wiki/Affymetrixhttp://en.wikipedia.org/wiki/Illumina_(company)http://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/DNA_microarray#Uses_and_typeshttp://en.wikipedia.org/wiki/DNA_microarray#Uses_and_typeshttp://en.wikipedia.org/wiki/DNA_microarray#Fabricationhttp://en.wikipedia.org/wiki/DNA_microarray#Microarrays_and_bioinformatics

  • 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