Molecular Biology applications of DNA dependant DNA polymerases

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Molecular Biology applications of DNA dependant
DNA polymerases
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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.

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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.

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Also produced recombinantly
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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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Uses of Klenow

• Filling in recessed 3' ends of DNA fragments

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Uses of Klenow

• Digesting away protruding 3' overhangs

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• 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

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• 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.

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• 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.

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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.

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• 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.

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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.

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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.

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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

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• 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.

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Sources of Commercially available RTs

• Moloney murine leukemia virus a single
  polypeptide
• Avian myeloblastosis virus composed of two
  peptide chains

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• 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.

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Uses

• cDNA
• RTPCR

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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.

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• 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)

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• 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.

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• 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.

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• 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.
• ?        

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• 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).

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