Scribd Logo
Upload

Welcome to Scribd!

  • 0 views

    BLS_307_05_edtd

    Uploaded by

    anicethmanyasi2

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd

    BLS_307_05_edtd

    Uploaded by

    anicethmanyasi2
    0 views21 pages

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    0 views21 pages

    BLS_307_05_edtd

    Uploaded by

    anicethmanyasi2

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    You are on page 1of 21

    Dolly the sheep (1997-2003)

    1. Animal and human cloning


    2. Gene cloning
    EgK;ekifaro07@gmail.com
    Department of Microbiology, Parasitology, and Biotechnology
    Animal and Human Cloning
    What is Gene Cloning?

    To "clone a gene" is to make multiple


    copies of it in vivo
    Why Clone DNA?
    • A particular gene can be isolated and its nucleotide sequence
    determined

    • Protein/enzyme/RNA function can be investigated

    • Mutations can be identified, e.g. gene defects related to


    specific diseases

    • Organisms can be ‘engineered’ for specific purposes, e.g.


    insulin production
    Gene Expression in Bacteria
    Gene encoding insulin
    RE1

    RE2

    RE1
    RE2
    RE1
    RE1

    RE2 RE2

    Ligation
    Gene Expression in Bacteria

    Transformation into E. coli cells

    Insulin
    What Do we Need for Gene Cloning?

    Target DNA Plasmid vector


    • Insert Ligation
    • Plasmid Vector
    • Ligation of the plasmid + Insert
    Transformation
    • Transformation
    Plasmid Vectors
    • Plasmids are small circles of DNA
    found in bacteria.

    • Plasmids replicate independently of


    the bacterial chromosome.

    • Pieces of foreign DNA can be


    added within a plasmid to create a
    recombinant plasmid.

    • Replicates a recombinant DNA


    Plasmid Vectors
    • Is a DNA molecule used for transferring foreign DNA
    fragments (genes) into host cells.

    • Circular DNA, capable of autonomous replication

    All plasmids contain:


    • 1. Origin of replication (ori)
    • 2. Selection marker (Ampicillin)
    • 3. Multiple Cloning Site (MCS)
    Insert – Target DNA

    1. PCR product

    2. Restriction Enzymes RE1

    RE1 RE1 RE2


    RE2
    RE2
    Ligation

    Ligation

    REs will produce ends that


    enable the gene to be spliced
    into a plasmid
    REs and DNA Ligase
    Ligation of the insert to the plasmid cut with only one enzyme
    1

    2
    2

    DNA ligase
    REs and DNA Ligase
    RE1
    RE1 RE2
    RE1 RE2
    RE2
    Ligation

    There is two possible outcomes


    Transformation
    Use of bacterial cells to amplify the DNA of interest

    Two main methods


    1.Chemical transformation – Chilling cells in the presence of Ca2+
    prepares the cell walls to become permeable to plasmid DNA.
    Cells are briefly heat shocked which causes the DNA to enter
    the cell.

    2.Electoporation- making holes in bacterial cells, by briefly


    shocking them with an electric field of 10-20kV/cm. Plasmid
    DNA can enter the cell through these holes.
    Possible Products of Transformation

    Plasmid + insert Plasmid without insert No plasmid


    Ampicillin resistant Ampicillin resistant No ampicillin resistance

    How can we differentiate between the bacteria containing


    plasmid+insert and the ones with the self ligated plasmid
    (no insert)?
    Cloning procedure
    Transformation

    + IPTG
    + X-Gal
    Lac Z gene
    Promotor LacZ gene
    RNA pol.

    LacZ mRNA
    Ribosome

    β-galactosidase

    X-gal BLUE colonies

    X-gal WHITE colonies


    Lac Z gene
    Promotor LacZ
    RNA pol.

    Promotor Operator LacZ


    RNA pol.
    Repressor IPTG
    IPTG IPTG

    Promotor Operator LacZ


    RNA pol.
    IPTG
    IPTG

    Β-galactosidase
    X-gal X + galactose
    Cells which produce ß-galactosidase form BLUE colonies; Cells without ß-galactosidase production form WHITE colonies.
    Screening Insert
    Promotor Operator T T LacZ
    pGEM

    X X X Without plasmid
    Plasmid without Insert
    X
    Plasmid +Insert

    WHITE colonies BLUE colonies


    Confirmatory experiment
    • Confirmation by digestion with RE and separation of the
    digestion products on agarose gel

    • A plasmid DNA will be purified from the bacteria cells.

    EcoRI Vector
    pGEM
    EcoRI

    Insert
    들어주셔서 감사합니다

    Kimchi

    Iced Sushi

    You might also like

    pFad - Phonifier reborn

    Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

    Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


    Alternative Proxies:

    Alternative Proxy

    pFad Proxy

    pFad v3 Proxy

    pFad v4 Proxy