ABE Workshop 2005July 15, 2006

1
Polymerase Chain Reaction (PCR)

• Kabi Neupane, Ph.D.
• Leeward Community College

2
Polymerase Chain Reaction

• Polymerase DNA polymerase
• DNA polymerase duplicates DNA
• Before a cell divides, its DNA must be duplicated
• Chain Reaction The product of a reaction is used
  to amplify the same reaction
• Results in rapid increase in the product

3
Polymerase Chain Reaction (PCR)

• PCR performs the chemistry of DNA duplication in
  vitro
• Numerous PCR applications make this process a
  staple in most biology laboratories
• Understanding properties of DNA polymerases
  helps understanding PCR

4
Discovery

• PCR was discovered by Kary Mullis
• On a long motorcycle drive
• Mentally visualized the process
• Nobel Prize in Chemistry
• 1993

5
DNA polymerase

• Duplicates DNA
• Necessary for reproduction of new cells
• More than one DNA polymerases exist in different
  organisms

6
Properties of DNA polymearse

• Needs a pre-existing DNA to duplicate
• Cannot assemble a new strand from components
• Called template DNA
• Can only extend an existing piece of DNA
• Called primers

7
Properties of DNA polymearse

• DNA polymerase needs Mg as cofactor
• Each DNA polymerase works best under optimal
  temperature, pH and salt concentration
• PCR buffer provides optimal pH and salt condition

8
Properties of DNA polymearse

• DNA strands are anti-parallel
• One strand goes in 5 ? 3
• The complementary strand is opposite
• DNA polymerase always moves in one direction
  (from 5 ? 3)

9
Properties of DNA polymearse

• DNA polymerase incorporates the four nucleotides
  (A, T, G, C) to the growing chain
• dNTP follow standard base pairing rule

10
Properties of DNA polymearse

• The newly generated DNA strands serve as template
  DNA for the next cycle
• PCR is very sensitive
• Widely used

11
Setting up a PCR Reaction

• Add template DNA and primers

• Add dNTPs

• Add DNA polymerase

12
Taq DNA polymerase

• Derived from Thermus aquaticus
• Heat stable DNA polymerase
• Ideal temperature 72C

13
Thermal Cycling

• A PCR machine controls temperature
• Typical PCR go through three steps
• Denaturation
• Annealing
• Extension

14
Denaturation

• Heating separates the double stranded DNA
• Denaturation
• Slow cooling anneals the two strands
• Renaturation

Cool
Heat
15
Annealing

• Two primers are supplied in molar excess
• They bind to the complementary region
• As the DNA cools, they wedge between two template
  strands
• Optimal temperature varies based on primer length
  etc.
• Typical temperature from 40 to 60 C

16
Extension

• DNA polymerase duplicats DNA
• Optimal temperature 72C

17
PCR Amplification
Exponential Amplification of template DNA
18
Typical PCR mix

• In a thin wall Eppendorf tube assemble the
  following

19
Applications

• Ubiquitous applications
• Revolutionized how we study biology
• Research
• Diagnostics
• Forensics