1. Polymerase Chain Reaction (PCR)
Lab 2

2. Today’s lab Principles of PCR The actual reaction Primer design
Limitations, troubleshooting and uses
Variations

3. DNA Structure
Sugar-Phosphate backbone

4. What Is PCR? In vitro technique
Amplification of specific DNA sequence between two regions of known sequence
Invented 1985

5. Overall Principle of PCR
DNA – 1 copy
Known sequence
Sequence of interest
PCR
Lots of copies

6. Why PCR? To ‘pull the needle out of the haystack’ Rapid & easy
Sensitive
Robust
Widespread applications

7. The Cycling Reactions Denature: ‘separate D.S DNA’
Anneal: ‘stick primers to S.S DNA’
Extend: ‘make new DNA from template’

8. Components of Reaction
Template DNA
Primers
DNA Polymerase
Taq
dNTPs
dTTP
dCTP
dGTP
dATP

9. Denaturation Temperature: 92-94C
Double stranded DNA melts single stranded DNA 3’ 5’
92C 5’ 3’ + 5’ 3’

10. Annealing
Temperature: ~50-70C (dependant on the melting temperature of the expected duplex)
Primers bind to their complementary sequences 5’ 3’
Forward primer
Reverse primer 5’ 3’

11. Extension Temperature: ~72C Time: 0.5-3min
DNA polymerase binds to the annealed primers and extends DNA at the 3’ end of the chain 5’ 3’
Taq 5’ 5’ 3’
Taq

12. Extension (2) 5’ 3’
Taq 5’ 5’
Taq 3’ 5’

13. Properties of Polymerase
Taq polymerase originally isolated from from Thermus aquaticus
Heat stable (half life of ~ 30min at 95C)
Taq DNA polymerase has no proof-reading function in 3’―5’ direction
Primer extension occurs at up to 100 bases/sec
Plateau is reached eventually

14. Products of Extension 5’ 3’
Taq 3’ 5’ 5’ 3’ 3’ 5’
Taq

15. Cycle Begins Again……… 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’

16. Products after 2 cycles 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’

17. Products after 3 cycles…..

18. Geometric Amplification
Timescale: e.g. 93C: 5 min
93C: 45 sec
53C: 45 sec
72C: 1 min
72C: 5 min
X 30

19. Polymorphisms Normal Sequence5’ 3’ 5’ 3’
Normal Sequence
Single base change at 3’ - primer won’t bind fully 5’ 3’ 5’ 3’
Sequence containing polymorphism

20. Dolan DNA learning centre

21. Primer Design Base composition: GC content 40-60% Length: ~18-24 bases
Melting temp (Tm) of 2 primers: shouldn’t differ by more than ~5C
Tm of amplification product: shouldn’t differ from Tm of primers by those of primers by more than ~10C

22. Primer Design (cont) 3’ terminal sequences critical
Cycling and buffer conditions adjusted for each primer pair
Complimentary sequences on pairs of primers to be avoided
Primer 1 3’ 3’
Primer Dimer
Primer 2

23. Not complimentary to DNA sequence – cannot bind
Primer Design (cont)
2 new primers generated
Self complimentary sequences on primers also to be avoided
Not complimentary to DNA sequence – cannot bind
Primer
Primer secondary structure

24. PCR Primer Design - Summary
PRIMER LENGTH - usually bases is optimal. A primer of 18 bases should, in theory, only hybridize in one position in a eukaryotic genome.
SELF-COMPLEMENTARITY -primer pairs should always be checked for complementarity. Complementarity at the 3’end can often lead to primer dimer formation. Complementary sequences within a single primer - especially if GC rich - can lead to hairpin loops or other secondary structures being formed. Commercially available computer programmes can be used to search for complementary sequences in primers so that these can be avoided.
SPECIFICITY - the 3’ terminal sequence of a primer is critical for PCR specificity and sensitivity. A run of 3 or more G or C bases at this position should be avoided ( can cause non-specific annealing ). Also T should not be used at the 3’ end as it is more prone to mis-priming than the other nucleotides. Bases at the 5’ end are less critical for primer annealing, so if a restriction enzyme site is to be introduced, it should be closer to this end.
G + C CONTENT - aim for 40% - 60%.

25. Things to try if PCR does not work
A) If no product ( of correct size ) produced:
1 Check DNA quality
2 Reduce annealing temperature
3 Increase magnesium concentration
4 Add dimethylsulphoxide ( DMSO ) to assay ( at around 10% )
5 Use different thermostable enzyme
6 Throw out primers - make new stocks
B) If extra spurious product bands present
1 Increase annealing temperature
2 Reduce magnesium concentration
3 Reduce number of cycles
4 Try different enzyme

26. Basic requirements for PCR reaction
1) DNA sequence of target region must be known. 2) Primers - typically bases in size. These can be readily produced by commercial companies. Can also be prepared using a DNA synthesizer 3) Thermo-stable DNA polymerase - eg Taq polymerase which is not inactivated by heating to 95C 4) DNA thermal cycler - machine which can be programmed to carry out heating and cooling of samples over a number of cycles.

27. STANDARD PCR REACTION Genomic DNA - 100ng ( approx )
Forward & reverse primers at μM concentration.
dATP, dCTP, dGTP, dTTP - each at 0.2mM conc.
Appropriate buffer containing magnesium.
Taq polymerase unit
Sterile H2O - to give required total reaction volume

28. STANDARD PCR METHOD
Prepare mix containing primers, dNTP’s, buffer and water sufficient for all reaction tubes or microtitre plate wells
Aliquot appropriate volume to each 0.2ml thin walled tube or plate well. (robotic system can be used with plates)
Add DNA to each tube - using a new tip for each sample
Add Taq polymerase - load tubes on PCR machine
If PCR machine does not have heated lid, 1 drop of mineral oil should be added to each tube before adding Taq. The Taq can be added with the tubes in the PCR machine block at 80C = hot start.
Start PCR programme

29. Example of PCR programme
Initial denaturation 95C for 5 mins
Thermo-cycle file - 30 cycles of
Denaturation : C for 30 secs
Annealing : 55C for 30 secs
Extension : 72C for 45 secs
Final extension 72C for 5 mins
Holding ( soak ) file usually 4C

30. Advantages of PCR
Small amount of DNA is required per test ng for PCR; 5-10μg for Southern blotting.
Result obtained more quickly - usually within 1 day for PCR; 5-10 days for Southern.
Usually not necessary to use radioactive material (32P) for PCR.
PCR is much more precise in determining the sizes of alleles - essential for some disorders.
PCR can be used to detect point mutations.

31. Checklist Is there a product formed?
Is the product of the correct size?
Is only one product formed?
1800bp

32. What can you do with PCR products?
Gel electrophoresis – presence/absence of product, size determination
Restriction endonuclease digestion
Sequencing

33. Applications
Amplify DNA for genetics applications, cloning, sequencing, mapping DNA segments, infectious disease diagnosis, sex determination, forensics, evolutionary biology, archaeology…….