Polymerase Chain Reaction

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Polymerase Chain Reaction

• PCR
• Salwa Qadri

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PCR

• 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
• 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

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PCR Animation

• http//www.maxanim.com/genetics/PCR/PCR.htm

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PCR Results

• These 3 steps of PCR result in exponential growth
  N 2t x (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 sample problem

• If you start with 30 copies of a target sequence
  and perform PCR for 45 cycles, then how many
  copies of the target sequence will you have at
  the end of the PCR amplification?

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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

• 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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In Lab.

• Pick Worms
• Lyse them in buffer
• Set up PCR
• Run a gel

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Wild-type vs. Knockout worms

• Use random mutation to mutagenize small number of
  worms
• Mutagenesis will produce a small deletion in any
  gene of interest
• PCR will allow the detection of deletion between
  primers
• Result knockout amplicon smaller in size
  compared with wild-type.

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Wild-type vs. Knockout worms

• PCR of smaller deletion amplicons is carried out
  more efficiently then large wild-types
• Work backward to identify subculture of worms
  with the deletion once a DNA pool with a deletion
  is identified
• Live animals carrying deletion mutation can be
  identified

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Real-Time PCR

• This procedure uses a fluorescence dye that will
  bind the DNA.
• Can view the increase in the amount of DNA as it
  is amplified.
• As the PCR reaction progresses, more DNA is
  produced and more fluorescence is detected
• Determine how much DNA was synthesized

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SYBR green is a fluorescence dye that binds
DNA
Adapted from Biorad.com
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Sybr green can be first detected at the
threshold cycle
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Can also use Ethidium bromide

• Ethidium bromide intercalates into the DNA
• Take DNA after different cycles and add ethidium
  bromide
• Compare to known standards