The Polymerase Chain Reaction

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The Polymerase Chain Reaction
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Polymerase Chain Reaction

• Developed by Kary Mullis in mid-1980s
• Revolutionary methods of gene analysis
• Enablement of production of enormous numbers of
  copies of a specified DNA sequence without the
  reliance to cloning

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PCR amplifies specific regions of DNA

• Exploitation of certain features of DNA
  replication
• Specifically, DNA polymerase which uses ssDNA as
  template for synthesis of a complementary new
  strand

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DNA review

• dsDNA strand is held together by weak hydrogen
  bonds between 2 of 4 nitrogenous bases that are
  complimentary to one another
• Adenine-Thymine
• Cytosine-Guanine
• Strands are anti-parallel, 5 ? 3 counter to 3
  ? 5

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PCR amplifies specific regions of DNA

• Utilization of a cyclical run of temperature
  fluctuations thereby allowing the newly created
  dsDNA to serve as a template for further daughter
  DNA strands
• ssDNA templates are produced by heating the dsDNA
  to be point of boiling (step 1)
• Requirement of DNA polymerase of a small section
  of dsDNA to initiate or prime the synthesis
  (step 2)

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Primers

• Selection of primers based on sequences
  juxtaposed on both sides of the target of
  interest
• Two primers should be designed one for each
  strand
• 18-24 bp
• Both primers should have similar Tm
• PurinePyrimidine content as close as 11
  (40-60)
• Avoiding primer-primer interactions
• Cooling of reaction after denaturation (step 1)
  to allow primers to bind or anneal to the ssDNA
  template
• Mg affects the annealing of the primer to the
  template DNA by stabilizing the primer-template
  interaction, it also stabilizes the replication
  complex of polymerase with template-primer

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Primers

• 5CTATGGATAAGATGAGAGGACTAAATGTATGAGAGAGTAATAGA
  GAG3
• 3GATACCTATTCTACTCTCCTGAT.TTACATACTCTCTCATTATC
  TCTC5
• step 1 (denature)
• 5CTATGGATAAGATGAGAGGACTAAATGTATGAGAGAGTAATAGA
  GAG3
• 3GATACCTATTCTACTCTCCTGAT.TTACATACTCTCTCATTATC
  TCTC5
• step 2 (anneal)
• 5CTATGGATAAGATGAGAGGACTAAATGTATGAGAGAGTAATAGA
  GAG3
• 3ACTCTCTCATTATCTC5
• 3GATACCTATTCTACTCTCCTGAT.TTACATACTCTCTCATTATC
  TCTC5
• 5GGATAAGATGAGAGG3
• step 3 (extend)
• 5CTATGGATAAGATGAGAGGACTAAATGTATGAGAGAGTAATAGA
  GAG3
• 3TACATACTCTCTCATTATCTC5
• 3GATACCTATTCTACTCTCCTGAT.TTACATACTCTCTCATTATC
  TCTC5

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Extension

• Use of taq DNA polymerase to synthesize new
  strand of DNA, complementary to template, that
  extends to a variable distance from the primer
  binding site, using the available separate
  deoxynucleotides in the reaction
• dATP, dCTP, dTTP, dGTP
• taq DNA polymerase has a higher error rate (no
  proof-reading 3' to 5' exonuclease activity)

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Cyclic reaction

• Post-extension involves the repeat of the entire
  process which causes the newly formed dsDNA to
  denature
• The newly synthesized dsDNA are separated into
  ssDNA, and 4 binding sites are available for
  primers to anneal
• Taq polymerase synthesizes the new complementary
  strand, however, the extension of these new
  strands is limited to the target sequence

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Amplification of target sequence only

• extension (step 3)
• denature (step 1)

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Amplification of target sequence only

• annealing (step 2)
• extension (step 3)

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Exponential Amplification
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(No Transcript)
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Resolving PCR products

• Polyacrylamide gel electrophoresis
• Higher resolving power separation of DNA
  molecules whos lengths differ by as little as .2
• Accommodation of higher quantities of DNA (up to
  10 ug) without resolution loss
• Higher purity of DNA can be extracted
• Nondenaturing PAGE gels for separation and
  purification of dsDNA fragments
• However, electrophoretic mobility is affected by
  base composition (in addition to size)
• Denaturing PAGE gels for separation and
  purification of ssDNA fragments
• Usually urea, formamide is used to suppress base
  pairing in nucleic acids
• Denatured DNA migrates at a rate independent of
  base composition

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Resolving PCR products

• Agarose gel
• Lower resolving power, but greater range of
  separation (200 bp to 50 kb)
• Buffers
• TAE (tris acetate) has low buffering capacity
  (ionic strength)
• TBE (tris borate) and TPE (tris phosphate) have
  higher buffering capacity

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Resolving PCR products

• Visualization of DNA via fluorescence
• Ethidium bromide
• Insertion between the bases of the DNA
• EtBRDNA complex displays an increase fluorescent
  yield compared to dye in free solution
• Yield is lower for ssDNA

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Metaphor Agarose

• Intermediate melting temperature
• 2x resolution capabilities of agarose
• Resolution of products differing in size by 2,
  in the range of 200 bp to 800 bp