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    618 Restriction Enzymes and Vectors

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    akshif Khan
    To construct a recombinant DNA molecule, one isolates DNA/RNA, isolates the gene of interest via PCR/RT-PCR, and cuts the DNA with restriction enzymes. The gene is then transferred into a vector like a plasmid using DNA ligase. The recombinant molecule is transformed into a host like E. coli and copies are produced. Gene expression is then checked.

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    © All Rights Reserved
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    618 Restriction Enzymes and Vectors

    Uploaded by

    akshif Khan
    10 views25 pages
    To construct a recombinant DNA molecule, one isolates DNA/RNA, isolates the gene of interest via PCR/RT-PCR, and cuts the DNA with restriction enzymes. The gene is then transferred into a vector like a plasmid using DNA ligase. The recombinant molecule is transformed into a host like E. coli and copies are produced. Gene expression is then checked.

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    3. 618 Restriction Enzymes and Vectors

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    To construct a recombinant DNA molecule, one isolates DNA/RNA, isolates the gene of interest via PCR/RT-PCR, and cuts the DNA with restriction enzymes. The gene is then transferred into a vector like a plasmid using DNA ligase. The recombinant molecule is transformed into a host like E. coli and copies are produced. Gene expression is then checked.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    10 views25 pages

    618 Restriction Enzymes and Vectors

    Uploaded by

    akshif Khan
    To construct a recombinant DNA molecule, one isolates DNA/RNA, isolates the gene of interest via PCR/RT-PCR, and cuts the DNA with restriction enzymes. The gene is then transferred into a vector like a plasmid using DNA ligase. The recombinant molecule is transformed into a host like E. coli and copies are produced. Gene expression is then checked.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 25

    How to construct a recombinant

    DNA molecule?
    • DNA/RNA isolation
    • Gene isolation by PCR/RT-PCR
    • Or Cutting of DNA molecule with the help of
    restriction enzymes
    • Transfer of DNA molecule into a suitable vector
    with the help of DNA ligases
    Transformation of recombinant molecule into
    suitable host like E. coli
    • Production of large number of copies of the
    recombinant molecule in the host
    • Checking the gene expression
    By Prof Amer Jamil
    Overexpression of proteins
    Restriction endonucleases

    • TYPE I: Restrict away from recognition


    site (~1000 bp away)

    •TYPE II: Restrict within recognition site

    •TYPE III: Restrict away from recognition


    site (~26 bp away)
    Type II Restriction endonucleases

    • Also called restriction enzymes


    • Occur naturally in bacteria
    • Hundreds are purified and available commercially
    • Named for bacterial genus, species, strain, and type

    Example: EcoRI

    Genus: Escherichia
    Species: coli
    Strain: R
    Restriction Endonuclease Specificity
    Restriction endonucleases
    recognize a specific DNA
    sequence, cutting ONLY at
    that sequence
    – They recognize 4-bp, 6-bp,
    8-bp palindromic sequences
    – The frequency of cuts
    lessens as the recognition
    sequence is longer
    – They cut DNA reproducibly
    in the same place
    4-7
    Restriction-Modification System
    • What prevents these
    enzymes from cutting up
    the host DNA?
    – They are paired with
    methylases (modification
    methylase)
    – Theses enzymes recognize,
    methylate the same site
    • Together they are called a
    restriction-modification
    system, R-M system
    • Methylation protects DNA,
    after replication the parental
    strand is already
    methylated 4-8
    Restriction enzymes conditions

    • Buffer systems (4 buffer system): pH and ionic


    strength
    • Optimal temperature
    • Star activity (non-specific cutting)
    • Enzyme activity (IU; umoles of substrate
    transformed into product in one minute).
    For restriction enzymes: Enzyme required to
    digest 1 ug double stranded DNA in 60 minutes)
    • Isoschizomers (two different enzymes
    recognizing the same sequence
    Gene is transferred to a suitable vector
    Vectors have selectable markers, origin of replication
    and increased copy numbers
    Commonly used vectors
    • Plasmids:
    – pUC18 (Expression vector)
    – pBR322 (cloning vector)
    • COSMID
    • Lambda vector
    • Agrobacterium tumefaciencs (used for plants)
    • YAC (yeast artificial chromosome)
    » And many more
    Plasmids pBR322 and pUC

    Three most important features of a plasmid:


    1. Origin of replication
    2. Selectable marker
    3. MCS (multiple cloning site)
    By Prof Amer Jamil
    Plasmid preparation for DNA ligation
    • The plasmid after digestion by
    restriction enzymes is
    dephosphorylated revents
    recircularization and re-ligation
    of linearized cloning vehicle
    DNA by removing phosphate
    groups from both 5´-termini
    • Dephosphorylation enzyme:
    • CIAP: calf-intestinal alkaline
    phosphatase
    • BAP: Bacterial alkaline
    phosphatase
    By Prof Amer Jamil
    Blunting incompatible sticky ends of DNA

    By Prof Amer Jamil


    Blunting incompatible sticky ends of DNA

    May remove overhangs:


    Exonuclease III removes nucleotide residues from the 3’-end of a DNA
    strand
    Bacteriophage ƛ exonuclease removes nucleotide residues from the 5’-end
    of a DNA strand

    By Prof Amer Jamil


    DNA ligation
    • T4 DNA ligase is used
    • It makes phosphodiester linkage (sealing
    the nick)
    Component Conc./amount in 20 uL
    T4 DNA ligase buffer 10X 2 uL
    Vector DNA (e.g. 4 kb) 50 ng (0.02 picomol)*
    Insert DNA (e.g. 1 kb) 37.5 ng (0.06 picomol)*
    T4 DNA ligase 1 uL
    Nuclease free water to 20 uL
    *molar ratio of 1:3 vector to insert
    By Prof Amer Jamil
    DNA Transformation
    •Heat shock or Electroporation
    •Competent cells: E. coli

    By Prof Amer Jamil


    Competent cells

    Ref. NEB
    DNA Transformation
    DNA Transformation
    Cell recovery period
    DNA Transformation
    Cell plating (with antibiotic)
    IPTG/X-gal in case of blue-white screening
    Selection of recombinant clones
    (blue-white screening)

    lac operon control


    A part of lacZ gene is in
    vector, and other part in
    chromosomal DNA
    Substrate: X-gal (dye)
    (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
    Inducer: IPTG
    (Isopropyl β-D-1-thiogalactopyranoside)
    By Prof Amer Jamil
    No vector in cell

    Killed by antibiotic
    such as ampicillin
    By Prof Amer Jamil
    IPTG + antibiotic

    By Prof Amer Jamil


    By Prof Amer Jamil

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