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    DNA Manipulative Enzymes

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    Zain Ul Abedien
    DNA manipulative techniques make use of purified enzymes to manipulate DNA molecules. These enzymes can be grouped into four classes: nucleases, ligases, polymerases, and modifying enzymes. Nucleases break DNA bonds, ligases form bonds, polymerases synthesize new DNA strands, and modifying enzymes alter DNA through addition or removal of chemical groups. Restriction endonucleases are important molecular scissors that cleave DNA at specific recognition sites and are used in genetic engineering.

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    DNA Manipulative Enzymes

    Uploaded by

    Zain Ul Abedien
    140 views17 pages
    DNA manipulative techniques make use of purified enzymes to manipulate DNA molecules. These enzymes can be grouped into four classes: nucleases, ligases, polymerases, and modifying enzymes. Nucleases break DNA bonds, ligases form bonds, polymerases synthesize new DNA strands, and modifying enzymes alter DNA through addition or removal of chemical groups. Restriction endonucleases are important molecular scissors that cleave DNA at specific recognition sites and are used in genetic engineering.

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    DNA manipulative techniques make use of purified enzymes to manipulate DNA molecules. These enzymes can be grouped into four classes: nucleases, ligases, polymerases, and modifying enzymes. Nucleases break DNA bonds, ligases form bonds, polymerases synthesize new DNA strands, and modifying enzymes alter DNA through addition or removal of chemical groups. Restriction endonucleases are important molecular scissors that cleave DNA at specific recognition sites and are used in genetic engineering.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    140 views17 pages

    DNA Manipulative Enzymes

    Uploaded by

    Zain Ul Abedien
    DNA manipulative techniques make use of purified enzymes to manipulate DNA molecules. These enzymes can be grouped into four classes: nucleases, ligases, polymerases, and modifying enzymes. Nucleases break DNA bonds, ligases form bonds, polymerases synthesize new DNA strands, and modifying enzymes alter DNA through addition or removal of chemical groups. Restriction endonucleases are important molecular scissors that cleave DNA at specific recognition sites and are used in genetic engineering.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 17

    DNA Manipulative

    Enzymes
    • The basis of recombinant DNA technology is the
    ability to manipulate DNA molecules in the test tube.
    • Almost all DNA manipulative techniques make use of
    purified enzymes.
    • Within the cell, these enzymes participate in
    essential processes such as DNA replication &
    transcription.
    Types of DNA
    Manipulative
    •
    Enzymes
    DNA manipulative enzymes can be grouped into four
    broad classes depending on the type of reaction that
    they catalyze:---
    1. Nucleases
    2. Ligases
    3. Polymerases
    4. Modifying enzymes
    1. Nucleases
    • Nucleases degrade DNA molecules by breaking the
    phosphodiester bonds that link one nucleotide to the
    next in a DNA strand.
    • Some nucleases are specific for DNA & some for
    RNA.
    • There are two different kinds of nucleases:-
    i. Exonucleases
    ii. Endonucleases
    i. Exonucleases
    • They remove nucleotides one at a time from the end
    of a DNA molecule.
    • The main distinction b/w different exonucleases lies
    in the number of strands that are degraded when a
    double-stranded molecule is attacked.
    A. Bal31:- It removes nucleotides from both strands of
    a double-stranded molecule.
    B. E.coli exonuclease III:- It removes nucleotides only
    from 3’ end of a double-stranded molecule.
    C. Lambda exonuclease:- It removes nucleotides only
    from 5’ end of a double-stranded molecule.
    ii. Endonucleases
    • They are able to break internal phosphodiester bonds
    within a DNA molecule.
    I. S1 endonuclease:- It cleaves only single-stranded DNA,
    including single-stranded nicks in mainly double-
    stranded DNA.
    II. Deoxyribonuclease I(DNase I):- It cleaves both single- &
    double- stranded DNA.
    • The special group of enzymes called Restriction
    Endonucleases cleave double-stranded DNA only at a
    specific recognition sites. It is also called molecular
    scissor.
    • These enzymes are found in bacteria & archaea, &
    provide a defense mechanism against invading viruses.
    2.
    Ligases
    • DNA ligase is used to seal the nicks that remain in the
    DNA molecule by forming a phosphodiester bond.
    • It is also called molecular glue.
    • E.g., T₄ polynucleotide ligase is prepared from E.coli
    cells infected with T₄ phage. It is involved in the
    replication of phage DNA & is encoded by the T₄
    bacteriophage.
    • The reaction requires energy which is provided by
    adding ATP to the reaction mixture.
    3. Polymerases
    • DNA polymerases are enzymes that synthesize a new
    strand of DNA complementary to an existing DNA or
    RNA template.
    • Four types of DNA polymerases are used in genetic
    engineering:---
    a. DNA Pol I
    b. DNA Pol II
    c. DNA Pol III
    d. Reverse Transcriptase
    4. DNA Modifying
    •
    Enzymes
    There are numerous enzymes that modify DNA molecules
    by addition or removal of specific chemical groups.
    • They are also called End-modification enzymes.
    • The most important are as follows:---
    I. Alkaline phosphatase:- It removes the phosphate
    group present at the 5’ terminus of a DNA molecule.
    II. T₄ polynucleotide kinase:- It adds phosphate groups to
    III. the 5’ terminus.
    Terminal deoxynucleotidyl transferase:- It adds one or
    more deoxyribonucleotides to the 3’ terminus of a single-
    & double-stranded DNA molecule. This is called Tailing.
    IV. Topoisomerases:- It introduce or remove supercoils
    from covalently closed circular DNA.

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