Polymerase Chain Reaction

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Polymerase Chain Reaction

• PCR

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PCR Apparatus
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PCR Apparatus
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PCR Apparatus
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Introduction

• PURPOSE to amplify a specific sequence
• WHY sometimes a DNA sample is so small that if
  you run it on a gel, you wont see it requires
  that you have MANY copies of the sequence

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PCR Amplification
Process
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PROCEDURE

• DNA is isolated
• Heat at 92ºC breaks H-bonds, DNA is ss
• Heat at 60ºC add primers anneal (2 diff)
• Heat at 70ºC Taq polymerase elongates 5' to 3'
• Repeat cycle about 30X
• Cycle 1 ? variable length strands
• Cycle 2 ? constant-length strands of target DNA
  
• 30 cycles ? 230 1B
• (Treat with endonucleases or probes)
• Run on a gel
• The machine is referred to as a thermocycler
• The technique is PCR

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PCR Procedure
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How is this useful?

• Once the DNA has been amplified, the quantity is
  large enough to be seen when run on a gel
• Bands run differently on gels because of the
  difference in the of bps
• This can be due to VNTR, LINES, SINES etc

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Application

• Heredity
• Amplify DNA from different family members to
  check heredity of a specific sequence
• Forensics and Paternity/Maternity Testing
• Amplify sample of DNA from crime scene to compare
  to suspects
• Presence of diseases
• Certain conditions can be diagnosed by the
  presence of a specific allelic seq or mutation
• Quantifying gene expression
• PCR with RNA req sufficient primers for
  accurate results
• Phylogeny (comparing species)
• Amplify DNA from a fossil and compare for seq
  similarity