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  • Recombinant DNA Technology Based On: Digest & Ligation

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    Recombinant DNA technology uses restriction enzymes to cut DNA at specific sites. Restriction enzymes cut DNA in different ways - Type I cuts nonspecifically, Type II cuts specifically producing sticky ends, and Type III cuts large fragments specifically. EcoRI is an example of a Type II restriction enzyme that produces sticky ends. Restriction enzymes are named based on the organism they are isolated from. The cut DNA can then be ligated back together to recombine genes in new ways. This technology has applications in producing proteins, developing gene therapies, and genetically modifying organisms.

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    Recombinant DNA technology uses restriction enzymes to cut DNA at specific sites. Restriction enzymes cut DNA in different ways - Type I cuts nonspecifically, Type II cuts specifically producing sticky ends, and Type III cuts large fragments specifically. EcoRI is an example of a Type II restriction enzyme that produces sticky ends. Restriction enzymes are named based on the organism they are isolated from. The cut DNA can then be ligated back together to recombine genes in new ways. This technology has applications in producing proteins, developing gene therapies, and genetically modifying organisms.

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    18 views10 pages

    Recombinant DNA Technology Based On: Digest & Ligation

    Uploaded by

    rengachen
    Recombinant DNA technology uses restriction enzymes to cut DNA at specific sites. Restriction enzymes cut DNA in different ways - Type I cuts nonspecifically, Type II cuts specifically producing sticky ends, and Type III cuts large fragments specifically. EcoRI is an example of a Type II restriction enzyme that produces sticky ends. Restriction enzymes are named based on the organism they are isolated from. The cut DNA can then be ligated back together to recombine genes in new ways. This technology has applications in producing proteins, developing gene therapies, and genetically modifying organisms.

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    of 10

    Recombinant DNA Technology

    based on
    Digest & Ligation
    Restriction Endonuclease/enzyme

    • H.O. Smith, K.W. Wilcox, &


    T.J. Kelley (1968)
    • Haemophilus influenzae
    (HindI)
    • Protective mechanism (R-M)
    • Methylase – protects genome
    • Endonuclease – against
    phage
    Types of restriction enzymes

    • Type I (R-M): nonspecific cuts


    • Type II (dimers): specific cuts
    • Type III (R-M): large fragments, specific cuts
    EcoRI (Palindromic – dimer)
    Naming of Restriction Enzyme
    •E Escherichia (genus)
    •co coli (species)
    •R RY13 (strain)
    •I First enzyme identified from this organism

    EcoRI Escherishia coli 5'GAATTC 5'---G AATTC---3'


    3'CTTAAG 3'---CTTAA G---5'
    BamHI Bacillus 5'GGATCC 5'---G GATCC---3'
    amyloliquefaciens 3'CCTAGG 3'---CCTAG G---5'
    AluI* Arthrobacter 5'AGCT 5'---AG CT---3'
    luteus 3'TCGA 3'---TC GA---5'
    TaqI Thermus aquaticus 5'TCGA 5'---T CGA---3‘
    3'AGCT 3'---AGC T---5'
    Plasmid Restriction Site Map
    Digest Reaction Table

    Single Digest Double Digest


    Reaction Reaction
    Plasmid (DNA):
    0.5 – 1 ug
    5 ul 5 ul
    Enzyme 1
    ___EcoR 1______ 2.5 ul 1.2 ul
    Enzyme 2
    ___BamH 1_____ None 1.3 ul
    Buffer # _2_
    5 ul 5 ul
    BSA
    1 ul 1 ul
    Sterile H2O
    36.5 ul 46.5 ul
    Total volume 50 ul 60 ul
    Ligation Reaction
    5’ 3’
    5’ 3’ GAT C C T A C T C AA C
    C A T A G C AA G
    G A T G A GT T G
    G T A T C G T T C C T AG 3’ 5’
    3’ 5’

    5’ 3’
    C A T A G C AA G GAT C C T A C T C AA C
    Fix hydrogen bonds
    G T A T C G T T C C T AGG A T G A G T T G
    3’ 5’

    5’ 3’
    C A T A G C AA G GAT C C T A C T C AA C
    Ligase fix phosphodiester
    G T A T C G T T C C T AGG A T G A G T T G bonds (backbones)
    3’ 5’

    5’ 3’
    C A T A G C AA G GAT C C T A C T C AA C
    Ligation completed
    G T A T C G T T C C T AGG A T G A G T T G
    3’ 5’
    Ligation Reaction Table

    V:I (1:1) V:I (1:3)


    Vector DNA
    (1ng)
    Insert DNA

    Ligase (5%)

    Ligase Buffer
    (1/10)
    Sterile H2O

    Total Volume
    Application of Recombinant
    DNA Technology
    • Efficient synthesis of
    important proteins
    • Medical therapeutic
    molecule synthesis
    • Possible genetic
    disorder therapy
    • Genetic modification of
    organisms with high
    economical values
    • Create new DNA/RNA
    sequences as research
    tool

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