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    Biotect - Unit Ii

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    Zeta Tuntun Gaming
    This document discusses gene cloning and its importance. It begins by outlining the early history of genetics from Mendel's work in 1900 to the discovery of DNA's structure in the 1950s. It then introduces gene cloning, describing how a gene can be inserted into a cloning vector which is used to introduce the gene into a host cell. The basic steps of gene cloning are outlined, including isolating the gene fragment, inserting it into a vector, transforming the recombinant DNA into host cells, and allowing the cells to replicate. Common vector types like plasmids, bacteriophages, and artificial chromosomes are described. Restriction enzymes are introduced as tools to cut DNA for insertion into vectors.

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    Biotect - Unit Ii

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    Zeta Tuntun Gaming
    35 views39 pages
    This document discusses gene cloning and its importance. It begins by outlining the early history of genetics from Mendel's work in 1900 to the discovery of DNA's structure in the 1950s. It then introduces gene cloning, describing how a gene can be inserted into a cloning vector which is used to introduce the gene into a host cell. The basic steps of gene cloning are outlined, including isolating the gene fragment, inserting it into a vector, transforming the recombinant DNA into host cells, and allowing the cells to replicate. Common vector types like plasmids, bacteriophages, and artificial chromosomes are described. Restriction enzymes are introduced as tools to cut DNA for insertion into vectors.

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    Original Title

    BIOTECT___UNIT II

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    This document discusses gene cloning and its importance. It begins by outlining the early history of genetics from Mendel's work in 1900 to the discovery of DNA's structure in the 1950s. It then introduces gene cloning, describing how a gene can be inserted into a cloning vector which is used to introduce the gene into a host cell. The basic steps of gene cloning are outlined, including isolating the gene fragment, inserting it into a vector, transforming the recombinant DNA into host cells, and allowing the cells to replicate. Common vector types like plasmids, bacteriophages, and artificial chromosomes are described. Restriction enzymes are introduced as tools to cut DNA for insertion into vectors.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    35 views39 pages

    Biotect - Unit Ii

    Uploaded by

    Zeta Tuntun Gaming
    This document discusses gene cloning and its importance. It begins by outlining the early history of genetics from Mendel's work in 1900 to the discovery of DNA's structure in the 1950s. It then introduces gene cloning, describing how a gene can be inserted into a cloning vector which is used to introduce the gene into a host cell. The basic steps of gene cloning are outlined, including isolating the gene fragment, inserting it into a vector, transforming the recombinant DNA into host cells, and allowing the cells to replicate. Common vector types like plasmids, bacteriophages, and artificial chromosomes are described. Restriction enzymes are introduced as tools to cut DNA for insertion into vectors.

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    of 39

    Subject: Pharmaceutical Biotechnology

    Paper Code: PT-619


    B. Pharm, 3rd Year, 6th Semester

    BY
    Miss Poulomi Biswas
    Assistant Professor
    EMINENT COLLEGE OF PHARMACEUTICAL TECHNOLOGY
    Moshpukur, Barbaria
    Barasat, 24 PGS(N), Kolkata
    Why Gene Cloning is Important?
    A century ago, Gregor Mendel :

    ❖ Basic assumption (each heritable property of an organism) is controlled


    by a factor (gene).

    ❖ In 1900, Mandel's law gave the birth of genetics.

    ❖ what these genes are and exactly how they work


    The Early Development of
    Genetics

    In 1903, Sutton, W. Proposed that


    genes reside on chromosomes
    In 1910, Morgan, TH

    ❖ Experimental backing on that --> development of the techniques for


    gene mapping (To establish the structure or structural details or
    location)
    ❖ By 1922, a comprehensive analysis of the relative positions of over 2000
    genes on the four chromosomes of the fruit fly. (Drosophilia
    melanogaster)

    ❖ In 1952, Hershey and Chase


    ❖ Experimental results were shown that DNA is the genetic material.
    ❖ Conventional idea : genes were made of protein
    ❖ In 1952-1966, Delbruck, Chargaff,
    Crick and Monod
    ❖ The structure of DNA was
    elucidated.
    ❖ The genetic code was cracked.
    ❖ The process of transcription and
    translation were described
    Introduction to Gene Cloning

    Let's suppose that we wish to construct a bacterium that produces human


    insulin.
    It might be thought that all that is required is to introduce the human insulin
    gene into its new host.

    Case A : The enzyme DNA polymerase, which makes copies of the DNA, does
    not initiate the process at random but at selected sites known as origins of
    replication.
    Replication: The process whereby a new daughter DNA molecule is synthesized
    from a parent DNA molecule.

    Case B : Recombinant DNA technology - Replication of the insulin gene in its


    new host by inserting the gene into a cloning vector. A cloning vector is simply
    a DNA molecule possessing an origin of replication and which can replicate in
    the host cell of choice.
    Basic Steps of Gene Cloning
    ❖ A fragment of DNA , containing the gene to be cloned, is inserted into a
    circular DNA molecule (vector)
    ❖ "Recombinant DNA molecule" or "Chimera"
    ❖ The vector acts as a vehicle that transports the gene into a host cell (usually,
    bacterium) possibly other types of living cell.
    ❖ Within the host cell the vector multiplies, producing numerous identical
    copies not only of itself but also of the gene that it carries.
    ❖ When the host cell divides, copies of the recombinant DNA molecule are
    passed to the progeny and further vector replication takes place.
    ❖ After a large number of cell divisions, a colony, or clone, of identical host
    cells is produced. Each cell in the clone contains one or more copies of the
    recombinant DNA molecule.
    ❖ The gene is cloned.
    Cloning Vectors
    ❖ Genetic vectors are vehicles for delivering foreign DNA into recipient
    cells.
    ❖ In molecular cloning, a vector is a DNA molecule used as a vehicle to
    artificially carry foreign genetic material into another cell, where it can be
    replicated and/or expressed.
    ❖ Vectors can replicate autonomously and typically include features to
    facilitate the manipulation of DNA as well as a genetic marker for their
    selective recognition.
    ❖ The different types of vectors available for cloning are plasmids,
    bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial
    chromosomes (YACs) and mammalian artificial chromosomes (MACs).
    ❖ The cloning vectors are limited to the size of insert that they can carry.
    ❖ Depending on the size and the application of the insert the suitable vector is
    selected for a particular purpose.

    ❖ Essential Characteristics of Cloning Vectors


    ❖ Regardless of the selection of a vector, all vectors are carrier DNA molecules.
    ❖ These carrier molecules should have few common features in general such as:
    ✓ It must be self-replicating inside host cell.
    ✓ It must possess a unique restriction site for RE enzymes.
    ✓ Introduction of donor DNA fragment must not interfere with replication property of
    the vector.
    ✓ It must possess some marker gene such that it can be used for later identification of
    recombinant cell (usually an antibiotic resistance gene that is absent in the host cell).
    ✓ They should be easily isolated from host cell.
    Plasmids
    ❖ Plasmids are extrachromosomal, self-replicating, usually circular, double-stranded DNA molecules
    found naturally in many bacteria and also in some yeasts
    ❖ The plasmid molecules can be present as 1 or 2 copies or in multiple copies (500-700) inside the host
    organism

    ❖ One of the earliest plasmid vectors to be constructed was pBR322

    ❖ This plasmid contains two different antibiotic resistance genes and recognition sites for several
    restriction enzymes. These vectors contain a region of the lacZ gene that codes for the enzyme β-
    galactosidase. This region also contains a polylinker and thus insertion of a foreign DNA into any of
    the restriction sites will result in an altered non-functional enzyme. The plasmid vectors described
    above can replicate only in E. coli. Many of the vectors used in eucaryotic cells are constructed such that
    they can exist both in the eukaryotic cell and E. coli. Such vectors are known as shuttle vectors.
    ❖ These vectors contain two types of origin of replication and selectable marker genes, one set which
    functions in the eukaryotic cells (e.g. yeast) and another in E. coli. An example of a shuttle vector is the
    yeast plasmid Yep. In the case of plants, a naturally occurring plasmid of the bacterium Agrobacterium
    tumefaciens called Ti plasmid has been suitably modified to function as a vector.
    Bacteriophage

    ❖ The viruses that infect bacteria are called bacteriophage. These are intracellular
    obligate parasites that multiply inside bacterial cell by making use of some or all of
    the host enzymes.

    ❖ Bacteriophages have a very high significant mechanism for delivering its genome
    into bacterial cell. Hence it can be used as a cloning vector to deliver larger DNA
    segments.

    ❖ Most of the bacteriophage genome is non-essential and can be replaced with


    foreign DNA.

    ❖ Using bacteriophage as a vector, a DNA fragment of size up to 20 kb can be


    transformed.
    Bacterial artificial chromosomes (BACs)
    • Bacterial artificial chromosomes (BACs) are simple plasmid which is designed to clone
    very large DNA fragments ranging in size from 75 to 300 kb.
    • BACs basically have marker like sights such as antibiotic resistance genes and a very
    stable origin of replication (ori) that promotes the distribution of plasmid after bacterial
    cell division and maintaining the plasmid copy number to one or two per cell.
    • BACs are basically used in sequencing the genome of organisms in genome projects
    (example: BACs were used in human genome project).
    Yeast artificial chromosomes (YACs)
    • YACs are yeast expression vectors.
    • A very large DNA fragments whose sizes ranging from 100 kb to 3000 kb can be cloned
    using YACs. Mostly YACs are used for cloning very large DNA fragments and for the
    physical mapping of complex genomes.
    • YACs have an advantage over BACs in expressing eukaryotic proteins that require post
    translational modifications. But, YACs are known to produce chimeric effects which make
    them less stable compared to BACs.
    Human artificial chromosomes (HACs)

    • Human artificial chromosomes (HACs) or mammalian artificial chromosomes


    (MACs) are still under development.
    • HACs are micro-chromosomes that can act as a new chromosome in a population of
    human cells.
    • HACs range in size from 6 to 10 Mb that carry new genes introduced by human
    researchers.
    • HACs can be used as vectors in transfer of new genes, studying their expression and
    mammalian chromosomal function can also be elucidated using these micro-
    chromosomes in mammalian system
    Restriction Endonucleases.

    In order to insert foreign DNA into a


    plasmid, use is made of special enzymes
    known as restriction endonucleases.
    These enzymes cut large DNA molecules
    into shorter fragments by cleavage at
    specific nucleotide sequences called
    recognition sites.
    These enzymes are highly specific
    deoxyribonucleases (DNases).
    Examples of Restriction Endonuclease
    Sticky end & Blunt end

    Sticky end : Some enzymes cut the two


    helices a few base pairs apart,
    generating two fragments with single-
    strand protrusions called sticky end.

    Blunt end (Flush end) : Some enzymes


    make a simple double-stranded cut in
    the middle of the recognition sequence.
    Making recombinant DNA

    ❖ Overview: Isolate DNA


    ❖ Cut with restriction enzymes
    ❖ Ligate into cloning vector transform recombinant DNA molecule into host cell
    ❖ each transformed cell will divide many, many times to form a colony of
    millions of cells, each of which carries the recombinant DNA molecule (DNA
    clone)

    A. Isolating DNA

    1. Crude isolation of donor (foreign) DNA is accomplished by isolating cells


    then disrupting lipid membranes with detergents destroying proteins with
    phenol or proteases degrading RNAs with RNase
    2. leaving DNA at the end
    Crude isolation of plasmid vector DNA is accomplished by an alkaline lysis procedure or by
    boiling cells which removes bacterial chromosomal DNA from plasmid DNA.
    3. To get purer DNA from either (1) or (2), crude DNA is
    a) Fractionated on a CsCl2 gradient
    b) Precipitated with ethanol
    c) Poured over a resin column that specifically binds DNA

    B. Cutting DNA
    1. DNA can be cut into large fragments by mechanical shearing.
    2. Restriction enzymes are the scissors of molecular genetics. Restriction enzymes (RE) are
    endonucleases that will recognize specific nucleotide sequences in the DNA and break the DNA
    chain at those points. A variety of RE have been isolated and are commercially available. Most
    cut at specific palindromic sites in the DNA (sequence that is the same on both antiparallel DNA
    strands). These cuts can be a staggered which generate “sticky or overhanging ends” or a blunt
    which generate flush ends.

    C. Joining DNA
    Once you have isolated and cut the donor and vector DNAs, they must be joined together. The
    DNAs are mixed together in a tube. If both have been cut with the same RE, the ends will match
    up because they are sticky. DNA ligase is the glue of molecular genetics that holds the ends of
    the DNAs together. DNA ligase creates a phosphodiester bond between two DNA ends.
    D. Amplifying the recombinant DNA

    To recover large amounts of the recombinant DNA molecule, it must be


    amplified. This is accomplished by transforming the recombinant DNA into a
    bacterial host strain. (The cells are treated with CaCl2 then DNA is added and
    Cells are heat shocked at 42 degree C and DNA goes into cell.)

    Once in a cell, the recombinant DNA will be replicated. When the cell divides,
    the replicated recombinant molecules go to both daughter cells which
    themselves will divide later. Thus, the DNA is amplified.

    DNA clone = A section of DNA that has been inserted into a vector molecule
    and then replicated in a host cell to form many copies.
    THERAPEUTIC AGENTS FOR HUMAN DISEASES

    Biotechnology is very useful for the production of several therapeutic products


    for treating human diseases. A selected list of rDNA derived therapeutic agents
    along with trade names and their uses in human are given below…..

    rDNA Product Trade name Application / Uses

    Insulin Humulin Diabetes

    Growth hormone Protropin/Humatrope Pituitary dwarfism

    Interferon Intron A Hairy cell leukemia


    Polymerase Chain Reaction

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