DNA Replication and the Polymerase Chain Reaction

1. DNA Replicationand thePolymerase Chain Reaction Timothy G. Standish, Ph. D.

2. History • The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention • A special DNA polymerase (Taq) is used to make many copies of a short length of DNA (100-10,000 bp) defined by primers • Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry

3. What PCR Can Do • PCR can be used to make many copies of any DNA that is supplied as a template • Starting with one original copy an almost infinite number of copies can be made using PCR • “Amplified” fragments of DNA can be sequenced, cloned, probed or sized using electrophoresis • Defective genes can be amplified to diagnose any number of illnesses • Genes from pathogens can be amplified to identify them (i.e., HIV) • Amplified fragments can act as genetic fingerprints

4. How PCR Works • PCR is an artificial way of doing DNA replication • Instead of replicating all the DNA present, only a small segment is replicated, but this small segment is replicated many times • As in replication, PCR involves: • Melting DNA • Priming • Polymerization

5. Origin of Replication 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ Initiation - Forming the Replication Eye

6. 3’ 5’ 5’ 3’ 3’ 5’ 3’ Primase 5’ Single-strand binding proteins Lagging Strand 5’ 5’ 3’ 5’ RNA Primers DNA Polymerase 5’ 3’ Helicase Leading Strand 5’ 3’ Extension - The Replication Fork Okazaki fragment

7. Function Enzyme Functions And Their Associated Enzymes • Ligase • Melting DNA • Helicase • SSB Proteins • Topisomerase • Polymerizing DNA • DNA Polymerase • Providing primer • Primase • Joining nicks

8. Components of a PCR Reaction • Buffer (containing Mg++) • Template DNA • 2 Primers that flank the fragment of DNA to be amplified • dNTPs • Taq DNA Polymerase (or another thermally stable DNA polymerase)

9. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers 72 oC Temperature 50 50 oC 0 T i m e 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ PCR

10. Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ 5’ 3’ PCR

11. Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ PCR Heat 5’ 3’

12. Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’ PCR

13. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’ PCR Heat Heat 5’

14. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ PCR

15. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ 5’ PCR Heat Heat

16. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ PCR

17. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ Fragments of defined length 5’ 5’ 5’ 5’ 5’ PCR

18. Number 1 2 4 8 16 32 64 0 Cycles 1 2 3 4 5 6 DNA Between The Primers Doubles With Each Thermal Cycle

19. Number of cycles 0 10 15 20 25 30 Size Marker More Cycles = More DNA

20. Theoretical Yield Of PCR Theoretical yield = 2n x y Where y = the starting number of copies and n = the number of thermal cycles If you start with 100 copies, how many copies are made in 30 cycles? 2nx y = 230x 100 = 1,073,741,824 x 100 = 107,374,182,400

21. Function PCR How The Functions Of Replication Are Achieved During PCR • N/A as fragments are short • Melting DNA • Heat • Polymerizing DNA • Taq DNA Polymerase • Providing primer • Primers are added to the reaction mix • Joining nicks

22. The End