Polymerase Chain Reaction

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Polymerase Chain Reaction

• PCR
• Thuy, Salwa, Hardik

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PCR

• PCR was invented by Dr. Kary Mullis in 1984. He
  was recognized and was awarded the Nobel Prize in
  1994.
• PCR allows for amplification of a small piece of
  DNA.
• PCR has become a popular alternative approach to
  cloning experiments.
• Some other applications of PCR are in forensics
  paternity/kinship testing, and the identification
  of human remains.

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PCR

• PCR requires a thermostable DNA polymerase
• The most common type of DNA polymerase is Taq
  polymerase
• It is obtained from the bacterium Thermus
  aquaticus which are found in hot springs
• Because the Taq polymerase is thermostable, it
  remains stable at near boiling temperatures
• 2 Oligonucleotides called primers are also
  included in the PCR reaction
• 4 deoxynucleotides- The subunits of DNA that are
  incorporated into the new DNA copies

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PCR machine

• The PCR mixture which contains DNA polymerase,
  buffer, deoxynucleotides, primers, and template,
  go through a cycle of varied temperatures
• 3 steps in a cycle
• -Denature
• -Anneal
• -Extend

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Steps in PCR

• Denature
• -Temperature is raised to near boiling (92ºC)
• -Template DNA is separated into two strands
• Anneal
• -Temperature is lowered (50º-65ºC)
• -Allows the left and right primers to base pair
  to their complementary sequences
• -Primers purpose is to bracket the DNA region to
  be amplified

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Steps in PCR

• Extend
• -Temperature is raised to 72ºC
• -Taq polymerase attaches at each priming site
• -Uses the nucleotides from the reaction mixture
  and attaches it to the growing strand these
  nucleotides are complementary to the target
  sequence
• -Nucleotides called dNTPs A, T, C or G
• dATP, dTTP, dCTP or dGTP

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PCR Results

• These 3 steps of PCR result in exponential growth
  N 2t (No) of the DNA template in the mixture
• Its typically repeated for 20-40 cycles
• But the product amount reaches a maximum after
  about 40 cycles due to depletion of the reaction
  components

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PCR- Problems

• Polymerase Errors
• Taq polymerase lacks 3 5 exonulcease activity
  
• Unwanted amplification of products
• -Contamination
• -Nonspecific primer annealing

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Applications of PCR

• After PCR the amplified products undergoes
  various experiments depending on the objective
• DNA fingerprinting used in forensics
• Paternity testing

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Application of PCR

• Identifying the dead
• - DNA analysis of 1000s of years DNA sample
• Gel electrophoresis
• Cloning experiments

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Cloning

• The amplified DNA is introduced into a plasmid
  vector
• Ligation
• Replication
• Detection

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• http//www.juliantrubin.com/encyclopedia/biochemis
  try/pcr.html
• http//www.thetech.org/genetics/images/ask/paterni
  tyTestFig3.gif