1. Polymerase Chain Reaction (PCR)
Chapter 5 Section 2
By: Kabi Neupane, Ph.D.
Edited by: Leah Spee

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: outside of the cell
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 Nobel Prize in Chemistry
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
Each DNA polymerase works best under optimal temperature, pH and salt concentration
PCR buffer provides optimal pH and salt condition

7. 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’) 5’ 3’ 3’ 5’

8. Properties of DNA polymearse
The newly generated DNA strands serve as template DNA for the next cycle
PCR is very sensitive
Widely used

9. Taq DNA polymerase Derived from Thermus aquaticus
Heat stable DNA polymerase
Ideal temperature 72C

10. Thermal Cycling A PCR machine controls temperature
Typical PCR go through three steps
Denaturation
Annealing
Extension

11. Denaturation Heating separates the double stranded DNA
Slow cooling anneals the two strands
Renaturation
Heat
Cool

12. Annealing Two primers are supplied.
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

13. What is a PRIMER?
Short chains of bases that hybridize to a target piece of DNA.
Primers determine the DNA fragment to be amplified in the PCR process.

14. Extension
Taq polymerase duplicates DNA
Optimal temperature 72C

15. PCR Amplification
Exponential Amplification of template DNA

16. Applications Ubiquitous applications
Revolutionized how we study biology
Research
Diagnostics
Forensics

17. PCR Animation