How PCR works

1. How PCR works • Cold Spring Harbor Animation • Animated .GIF files • Blackboard

2. Review: The structure of DNA Unzipping Antiparallel Strands

3. How PCR works • Cold Spring Harbor Animation PCR.EXE

4. How PCR works • Animated .GIF #1

5. How PCR works • Animated .GIF #2

6. How PCR works • Blackboard (Dave attempts to draw this out!)

7. PCR Reaction Components • Water • Buffer • DNA template • Primers • Nucleotides • Mg++ ions • DNA Polymerase

8. PCR Reaction: Water • Water • The medium for all other components.

9. PCR Reaction: Buffer • Water • Buffer • Stabilizes the DNA polymerase, DNA, and nucleotides • 500 mM KCl • 100 mM Tris-HCl, pH 8.3 • Triton X-100 or Tween

10. PCR Reaction: Template DNA • Water • Buffer • DNA template • Contains region to be amplified • Any DNA desired • Purity not required • Should be free of polymerase inhibitors

11. PCR Reaction: Primers • Water • Buffer • DNA template • Primers • Specific for ends of amplified region • Forward and Reverse • Annealing temps should be known • Depends on primer length, GC content, etc. • Length 15-30 nt • Conc 0.1 – 1.0 uM (pMol/ul)

12. PCR Reaction: Nucleotides • Water • Buffer • DNA template • Primers • Nucleotides • Added to the growing chain • Activated NTP’s • dATP, dGTP, dCTP, dTTP • Stored at 10mM, pH 7.0 • Add to 20-200 uM in assay

13. PCR Reaction: Magnesium • Water • Buffer • DNA template • Primers • Nucleotides • Mg++ ions • Essential co-factor of DNA polymerase • Too little: Enzyme won’t work. • Stabilizes the DNA double-helix • Too much: DNA extra stable, non-specific priming, band smearing • Used at 0.5 to 3.5 uM in the assay

14. PCR Reaction: Polymerase • Water • Buffer • DNA template • Primers • Nucleotides • Mg++ ions • DNA Polymerase • The enzyme that does the extension • TAQ or similar • Heat-stable • Approx 1 U / rxn

15. PCR Reaction Components • Water • Buffer • DNA template • Primers • Nucleotides • Mg++ ions • DNA Polymerase

16. A Typical PCR Reaction Component ml Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (10mM each) 1.0 PrimerFWD (25 pmol/ml) 1.0 PrimerREV 1.0 DNA Polymerase 0.5 DNA Template 1.0 Total Volume 50.0

17. A Simpler PCR Reaction Component ml Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (10mM each) 1.0 PrimerFWD (25 pmol/ul) 1.0 PrimerREV 1.0 DNA Polymerase 0.5 DNA Template 1.0 Total Volume 50.0 • Component ml • PREMIX 24.0 • Buffer • MgCl2 • dNTP’s • DNA Polymerase • “Enhancers” • Sterile Water • Primers FWD+Rev 1.0 DNA Template 25.0 • Total Volume 50.0 PREMIXES CAN REDUCE THE NUMBER OF ITEMS ADDED TO THE MIX

18. Using a PCR Mastermix Component 1X(ml) 20X(ml) Sterile Water 38.0 760 10X PCR Buffer 5.0 100 MgCl2 (50mM) 2.5 50 dNTP’s (10mM each) 1.0 20 PrimerFWD (25 pmol/ul) 1.0 20 PrimerREV 1.0 20 DNA Polymerase 0.5 10 DNA Template 1.0 -- Total Volume 50.0 980 Aliquot 49 ml Add DNA as last step

19. Typical Thermal Cycler Conditions 1. Initial Denaturation 95°C 4 min 2. DNA Denaturation 95°C 1 min 3. Primer Annealing 65°C 1 min 4. Primer Extension 72°C 1 min 5. Go to step #2, repeat 29 more times 6. Hold at 4 C 7. End

20. Finally: Some Mis-Uses (malas aplicaciones) of PCR • Since PCR is extremely sensitive, it is highly prone to giving “false positive” results. • Forensic and medical applications use strict protocols to minimize chance of errors. • Universities, working without standardized protocols, have frequently made incredible “discoveries”, only to find out later that it was all due to PCR artifacts. • Ancient DNA • GM Crops

21. MisUses of PCR: Ancient DNA According to Richard Thomas and his colleagues at London's Natural History Museum, writing in the August issue of Trends in Ecology and Evolution: 'It is highly unlikely that geologically ancient DNA survives in any fossil material so far studied.' The researchers spent three years attempting to extract DNA from insects entombed in amber. 'We worked with many more samples than the total number of published reports of success,' says Thomas. The result: complete failure. Not a single strand of prehistoric DNA to be seen. When an organism dies, its tissues immediately begin to break down, creating a soup of enzymes, water and oxidative molecules that cut DNA strands into smaller and smaller fragments. As time passes, the amount of DNA remaining in the tissue steadily diminishes, and eventually disappears entirely. http://www.newscientist.com/hottopics/dinosaurs/backfrom.jsp

22. MissUses of PCR: GM Crops An intense scientific debate has opened up over whether genetically modified (GM) crops in Mexico have contaminated wild maize (corn). Last November, the magazine Nature published data from two authors who said they had detected DNA from GM plants in wild crops growing in a remote area. The criticisms of Quist and Chapela concentrate on their use of a method known as i-PCR, inverse polymerase chain reaction - a technique used to amplify samples to sufficient levels so that they can be more easily studied. ...the conclusion of the original paper "seems to be based on an artefact arising from the i-PCR [Quist and Chapela] used... http://news.bbc.co.uk/hi/english/sci/tech/newsid_1911000/1911535.stm

23. “End” of PCR Introduction