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Molecular Biology applications of DNA dependant DNA polymerases

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The holoenzyme DNA polymerase I is no longer as frequently used. ... Synthesis of double-stranded DNA from single-stranded templates ... – PowerPoint PPT presentation

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Title: Molecular Biology applications of DNA dependant DNA polymerases


1
Molecular Biology applications of DNA dependant
DNA polymerases
2
E. coli DNA polymerase I
  • The E. coli DNA polymerase I is a DNA-dependent
    DNA polymerase that possesses both 3' - 5' and
    5' - 3' exonuclease activities. It is a
    single-chain protein with a mass of about 109,000
    Da that requires magnesium as a cofactor. Each of
    its three enzymatic activities are encapsulated
    into distinct domains of the holoenzyme, such
    that proteolytic deletions can be generated that
    lack one or more of the activities.
  • DNA polymerase I was used frequently in the early
    days of recombinant DNA technology for
    radiolabeling DNA and synthesizing cDNA. However,
    other enzymes have proven to be more effective
    for these purposes, including a proteolytic
    fragment of DNA polymerase I called Klenow
    fragment and T4 DNA polymerase. The holoenzyme
    DNA polymerase I is no longer as frequently used.

3
Large (Klenow) Fragment of E. coli DNA Polymerase
I
  • The 5' - 3' exonuclease activity of E. coli's
    DNA polymerase I is removed.
  • Exposure of DNA polymerase I to the protease
    subtilisin cleaves the molecule into two
    fragments
  • 5' - 3' exonuclease activity is on one fragment
  • The other is called the Klenow fragment. The
    Klenow fragment of DNA polymerase I has DNA
    polymerase and 3' - 5' exonuclease activities,
    and is widely used in molecular biology.

4
Also produced recombinantly
5
Uses of Klenow
  • Synthesis of double-stranded DNA from
    single-stranded templates
  • To use Klenow to synthesize a complementary
    strand of DNA, one simply mixes single-stranded
    template (usually denatured double-stranded DNA),
    primers and the enzyme in the presence of an
    appropriate buffer

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7
Uses of Klenow
  • Filling in recessed 3' ends of DNA fragments

8
Uses of Klenow
  • Digesting away protruding 3' overhangs

9
  • The 3' - 5' exonuclease activity of Klenow will
    digest away the protruding overhang. Removal of
    nucleotides from the 3' ends will continue, but
    in the presence of nucleotides, the polymerase
    activity will balance the exonuclease activity,
    yielding blunt ends. This reaction is more
    efficiently conducted with T4 DNA polymerase,
    which has much more potent exonuclease activity

10
  • Preparation of radioactive DNA probes
    Radioactive nucleotides can be incorporated into
    the DNA fragment. Klenow fragment is used
    frequently to prepare DNA that is labeled with
    radionuclides or other markers.

11
  • In some situations, the 3' - 5' exonuclease
    activity of Klenow fragment is either undesirable
    or not necessary. By introducing mutations in the
    gene that encodes Klenow, forms of the enzyme can
    be expressed that retain polymerase activity, but
    lack any exonuclease activity. These forms of the
    enzyme are usually called exo- Klenow fragment.

12
T4 DNA Polymerase
  • T4 is a bacteriophage of E. coli. The activities
    of T4 DNA polymerase are very similar to Klenow
    fragment of DNA polymerase I - it functions as a
    5' - 3' DNA polymerase and a 3' - 5'
    exonuclease, but does not have 5' - 3'
    exonuclease activity.

13
  • In general, T4 DNA polymerase is used for the
    same types of reactions as Klenow fragment,
    particularly in blunting the ends of DNA with 5'
    or 3' overhangs. There are however, two
    differences between the two enzymes that have
    practical significance
  • The 3' - 5' exonuclease activity of T4 DNA
    polymerase is roughly 200 times that of Klenow
    fragment, making it preferred by many
    investigators for blunting DNAs with 3'
    overhangs.
  • While Klenow fragment will displace downstream
    oligonucleotides as it polymerizes, T4 DNA
    polymerase will not.

14
T7 DNA Polymerase
  • The DNA polymerase of T7 bacteriophage has DNA
    polymerase and 3' - 5' exonuclease activities,
    but lacks a 5' - 3' exonuclease domain. It is
    thus very similar in activity to Klenow fragment
    and T4 DNA polymerase.
  • The claim to fame for T7 DNA polymerase is its
    processivity. That is to say, the average length
    of DNA synthesized before the enzyme dissociates
    from the template is considerably greater than
    for other enzymes. Due to this talent, the
    principle use of T7 DNA polymerase is in DNA
    sequencing by the chain termination technique.
  • T7 DNA polymerase can be chemically treated or
    genetically engineered to abolish its 3' - 5'
    exonuclease activity. These forms of the enzyme
    are marketed under the name Sequenase and
    Sequenase 2.0, and are widely used for DNA
    sequencing reactions.

15
Thermostable DNA Polymerases
  • Thermus aquaticusTaq
  • The thermophilic DNA polymerases, like other DNA
    polymerases, catalyze template-directed synthesis
    of DNA from nucleotide triphosphates. A primer
    having a free 3' hydroxyl is required to initiate
    synthesis and magnesium ion is necessary. In
    general, they have maximal catalytic activity at
    75 to 80C, and substantially reduced activities
    at lower temperatures. At 37C, Taq polymerase has
    only about 10 of its maximal activity.

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Error rate
  • polymerases lacking 3'-5' exonuclease activity
    generally have higher error rates than the
    polymerases with exonuclease activity. The total
    error rate of Taq polymerase has been variously
    reported between 1 x 10-4 to 2 x 10-5 errors per
    base pair. Pfu polymerase appears to have the
    lowest error rate at roughly 1.5 x 10-6 error per
    base pair, and Vent is probably intermediate
    between Taq and Pfu.

18
Reverse Transcriptases
  • Reverse transcriptase is a common name for an
    enzyme that functions as a RNA-dependent DNA
    polymerase. They are encoded by retroviruses,
    where they copy the viral RNA genome into DNA
    prior to its integration into host cells

19
  • Reverse transcriptases have two activities
  • DNA polymerase activity In the retroviral life
    cycle, reverse transcriptase copies only RNA,
    but, as used in the laboratory, it will
    transcribe both single-stranded RNA and
    single-stranded DNA templates with essentially
    equivalent efficiency. In both cases, an RNA or
    DNA primer is required to initiate synthesis.
  • RNase H activity RNase H is a ribonuclease that
    degrades the RNA from RNA-DNA hybrids, such as
    are formed during reverse transcription of an RNA
    template. This enzyme functions as both an
    endonuclease and exonuclease in hydrolyzing its
    target.

20
Sources of Commercially available RTs
  • Moloney murine leukemia virus a single
    polypeptide
  • Avian myeloblastosis virus composed of two
    peptide chains

21
  • The murine leukemia virus enzyme has very weak
    RNase H activity compared to the avian
    myeloblastosis enzyme, which makes it the clear
    choice when trying to synthesize complementary
    DNAs for long messenger RNAs.

22
Uses
  • cDNA
  • RTPCR

23
Other DNA modifying enzymes
  • Nuclease BAL-31
  • This is an exonuclease (starts at the termini and
    works inward) which will degrade both 3' and 5'
    termini of double stranded DNA. It will not make
    internal cleavages ("nicks"), however, it will
    degrade the ends of DNA at existing internal
    "nicks" (which create both 3' and 5' termini).
  • The degradation of termini is not coordinated,
    meaning that the product is not 100 blunt ended
    (even though the original duplex may have been
    blunt ended).
  • Such "ragged" ends can be made blunt by filling
    in and chewing back by a suitable polymerase
    (e.g. T4 DNA polymerase). The unit definition is
    1 unit is amount of enzyme required to remove 200
    base pairs from each end of duplex DNA in 10
    minutes at 30 C.

24
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25
  • Deoxyribonuclease I (DNAse I) from Bovine
    pancrease
  • This enzyme hydrolyzes duplex or single DNA
    strands preferentially at the phosphodiester
    bonds 5' to pyrimidine nucleotides
  • In the presence of Mg2 ion, DNAse I attacks each
    strand independently and produces nicks in a
    random fashion (useful for nick-translation)
  • In the presence of Mn2 ion DNAse I cleaves both
    strands of DNA at approximately the same position
    (but leaving "ragged" ends)

26
  • T4 DNA ligase
  • Isolated from bacteriophage T4.
  • Will ligate the ends of duplex DNA or RNA.
  • This enzyme will join blunt-end termini as well
    as ends with cohesive (complementary) overhanging
    ends (either 3' or 5' complementary overhangs).
  • This enzyme will also repair single stranded
    nicks in duplex DNA, RNA or DNA/RNA duplexes.
    Requires ATP as a cofactor.

27
  • Ligases
  • Ligases catalyze the formation of a
    phosphodiester bond between juxtaposed 5'
    phosphate and 3' hydroxyl termini of nucleotides
    (potentially RNA or DNA depending on the ligase).
  • In a sense, they are the opposite of restriction
    endonucleases, but they do not appear to be
    influenced by the local sequence, per se.
  • Ligases require either rATP or NAD as a
    cofactor.
  • DNA ligases are involved in fundamental processes
    such as DNA replication and repair, they are also
    used in molecular cloning.

28
  • Calf intestinal phosphatase (CIP)
  • Catalyzes the removal of 5' phosphate groups from
    RNA, DNA and ribo- and deoxyribo- nucleoside
    triphosphates (e.g. ATP, rATP).
  • CIP treated duplex DNA cannot self ligate.
  • ?        

29
  • Alkaline Phosphatase, Shrimp Heat-labile for
    easier dephosphorylation of DNA prior to cloning
  • Shrimp alkaline phosphatase (SAP) will remove the
    5' phosphate from DNA fragments (with 5'
    protruding, 3' protruding, or blunt ends) and
    RNA. SAP requires no dilution and is easily heat
    denatured (15 minutes, 65C). SAP eliminates the
    need for intermediate phenol/chloroform
    extraction steps between dephosphorylation and
    cloning. Thus, SAP allows dephosphorylation to be
    performed in the same reaction tube as
    restriction enzyme digestion and subsequent
    cloning (or end-labeling).

30
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