Primers and Polymerase Chain Reaction PCR

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Primers and Polymerase Chain Reaction (PCR)

• Shakesha Smith
• Denisse Iacobucci

Biocomputing Presentation February 3, 2009
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What is PCR and why is it important?

• PCR utilizes a DNA polymerase to amplify a target
  sequence of DNA exponentially.
• Importance?
• -DNA cloning for sequencing
• -Analysis of genes
• -Diagnosis of hereditary and infectious diseases
• -Genetic fingerprinting
• Benefit Results in several copies of the target
  sequence in a few hours
• Limitation Primer lacks 3 to 5 exonuclease
  activity

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What is needed to do PCR?

• DNA template The DNA to be amplified
• Primers Complementary to the 3 and 5 ends of
  the DNA template
• DNA polymerase Synthesizes the DNA
• dNTPs Building blocks which the polymerase uses
  to synthesize the DNA
• Buffer solution Creates the optimal environment
  for the reaction to occur
• MgCl2 Activates the polymerase

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Primer

• Contains specific nucleotides for replication
  region
• Initiates DNA replication
• Anchors DNA Polymerase
• PCR
• DNA clone

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Use in PCR

• Start DNA replication reaction
• Makes directed sequencing reactions
• Amplification
• 4 cycles
• 8 copies

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The Steps of each cycle of PCR

• Step 1 Denaturation
• Complimentary strands are separated
• Occurs at 95C
• Step 2 Annealing
• Left and right primers anneal to their
  complimentary sequence of DNA
• Occurs at 55C - 60C
• Step 3 Extension
• DNA polymerase extends the strands of DNA
  adding complimentary nucleotides using the dNTPs
• Occurs at 72C
• - The original DNA strand has been replicated.
  After each cycle, the DNA increases
  exponentially!

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References

• http//www.dnalc.org/ddnalc/resources/pcr.html
• http//www.pcrstation.com/learn-about-pcr/
• http//en.wikipedia.org/wiki/Polymerase_chain_reac
  tion
• Valverde, JR, EMBnet/CNB Internet. Primer
  Selection cited 2009 February 1 about 6
  screens. Available from http//www.es.embnet.org
  /Doc/ECJ/ECJ-1999-01/coursecnb/primer/index.html.