1. Release date: November 2015 © Copyright by Amplyus LLC, all rights reserved DNA amplification and analysis: miniPCR TM Food Safety Lab Science for everyone, everywhere

2. 1 © Copyright by Amplyus LLC, all rights reserved Welcome Our goals for today: Review DNA amplification theory Solve a public health problem using DNA analysis

3. 2 © Copyright by Amplyus LLC, all rights reserved Polymerase Chain Reaction (PCR) A process that identifies and copies (amplifies) a specific piece of DNA in a biological sample Complex DNA sample Amplified DNA (Billions of copies) Region of interest  Sequencing  Genetic risk  Pathogen detection  Drug development  Crop modification  Forensic analysis  Etc. Applications

4. 3 © Copyright by Amplyus LLC, all rights reserved PCR relies on DNA’s unique structure Source: US National Library of Medicine, NIH, Thinkquest DNA: a double helix......held together by base complementarity

5. 4 © Copyright by Amplyus LLC, all rights reserved How PCR works: 3 steps to copy DNA Denaturation 1 94°C Annealing 2 50-60°C Primer 2 Primer 1 Extension 3 72°C Taq DNA polymerase dNTPs

6. 5 © Copyright by Amplyus LLC, all rights reserved How PCR works: repeat the cycle DenaturationAnnealingExtension DNA + primersdenatured DNADNA + copy 94° C 50-60° C 72° C Repeat x ~25-30 cycles Single molecule~1B copies

7. 6 © Copyright by Amplyus LLC, all rights reserved PCR makes DNA visible (and useful)

8. 7 © Copyright by Amplyus LLC, all rights reserved PCR Text PCR is central to biomedical applications Forensics Food and agriculture Consumer genomics Molecular diagnostics Personalized medicine Human evolution Text

9. 8 © Copyright by Amplyus LLC, all rights reserved Outbreak of E. coli in ground beef  Dozens of victims nationwide Ongoing USDA investigation  Search for culprit meat processing plant  Test DNA samples for E. coli O157:H7 Use of core biotechnology techniques  PCR  Restriction digest  DNA electrophoresis Demonstration of real-world impact  DNA in public health / surveillance  Biotechnology in food industry We will use DNA technology to solve a real-world problem

10. 9 © Copyright by Amplyus LLC, all rights reserved Scientists work hard at detecting foodborne pathogens Most Escherichia coli are harmless and live in our gut Pathogenic strains can be serotyped by O/H antigens 0157:H7 strains can cause severe hemorrhagic diarrhea Complications can cause kidney damage and eventually, death Bacterial culture and antigen detection can be slow and fairly insensitive

11. 10 © Copyright by Amplyus LLC, all rights reserved Outbreaks are a serious public health concern Outbreaks of E. coli O157:H7 occur Every year A single outbreak can last Several months O157:H7 infection results in 2,100 hospitalizations annually A single outbreak can lead to recalls of millions of pounds of food PCR analysis can serve to detect and stop the spread of infection Why is PCR useful? PCR is ideal for fast detection of low concentrations of organisms e.g., 15 cells per g Source: http://www.cdc.gov/ecoli/general/index.html#what_shiga and http://www.cdc.gov/ecoli/outbreaks.html

12. 11 © Copyright by Amplyus LLC, all rights reserved In the news

13. 12 © Copyright by Amplyus LLC, all rights reserved How biotechnology can help fliC locus (non-pathogenic ) fliC fliC locus (pathogenic O157:H7 ) XmnI site Pathogenic E.coli gene differs from non-pathogenic E.coli Single nucleotide polymorphism (SNP) in fliC gene e.g. AAATTT changes to AAGTTT The SNP creates a restriction site in the fliC gene in E.coli O157:H7 SNP

14. 13 © Copyright by Amplyus LLC, all rights reserved fliC Lab plan Template DNA PCR amplification Gel electrophoresis Meat plant A non-pathogenic Meat plant B O157:H7 Restriction digest 400bp 150+250bp fliC XmnI site

15. 14 © Copyright by Amplyus LLC, all rights reserved 1.Template DNA to be amplified 2.Pair of DNA primers 3.DNA polymerase 4.dNTPs 5.Buffer to maintain pH and provide Mg 2+ 6.Thermal cycler What goes in a PCR reaction FWD primer REV primer Taq A A A A A A A T T T T G T T C C C C C C C G G G G G G G

16. 15 © Copyright by Amplyus LLC, all rights reserved A.DNA sample from meat packing B.plant “A” B.DNA sample from meat packing plant “B” D.Control DNA from non-pathogenic E.coli H.Control DNA from pathogenic I.E.coli Setting up your PCR reactions Label 4 PCR tubes per group Tube “A”Tube “B” Tube “P”Tube “NP” FIELD SAMPLES USDA “REFERENCE LAB”

17. 16 © Copyright by Amplyus LLC, all rights reserved Prepare 4 PCR tubes (200µL tubes) DNA sample A 15 µL 10 µL 5 µL A DNA sample B B Control P DNA P Control NP DNA NP 2X EZ PCR Master mix (EZ PCR Master Mix: PCR Buffer + Mg 2+ + Taq + dNTPs) 3X Food Safety Primer mix (Forward and Reverse primers) Also add your initials to side of tube

18. 17 © Copyright by Amplyus LLC, all rights reserved Programming PCR parameters Initial denaturation:94°C 30 seconds Denaturation:94°C 5 seconds Annealing57°C 5 seconds Extension72°C 5 seconds x25 cycles (if prioritizing speed) OR x30 cycles (if prioritizing robustness of results) Final extension72°C 30 seconds

19. 18 © Copyright by Amplyus LLC, all rights reserved Monitoring PCR amplification What is happening to DNA molecules at each step? Denaturation Annealing Extension Why do we need to add an enzyme (Taq polymerase)? What temperature is optimal for most enzymes? What makes Taq unique? How many more molecules of DNA will we have with each PCR cycle? And at the end of the entire PCR reaction? We call this exponential amplification How will we know which meat processing plant is behind the outbreak? Which caveats should the USDA investigators consider?

20. 19 © Copyright by Amplyus LLC, all rights reserved Quiz: Which of these are NOT characteristics of PCR primers?  A. Short synthetic oligonucleotide  B. Typically 18-25 bases in length  C. Double stranded DNA  D. Unique homology to the DNA template  E. Sequence with ~50% G:C content

21. 20 © Copyright by Amplyus LLC, all rights reserved Set up restriction digest (XmnI) PCR product ABPNP 15 µL AXBXPXNPX Restriction digest (incubate at 37°C) 1 µL Restriction enzyme (XmnI restriction endonuclease) +

22. 21 © Copyright by Amplyus LLC, all rights reserved Incubate 10 minutes at 37°C

23. 22 © Copyright by Amplyus LLC, all rights reserved Next step: visualize the DNA fragments amplified by PCR 1. Pour an agarose gel2. Load the PCR products 4. Visualization in a transilluminator3. Electrophoresis e- - Pole + Pole

24. 23 © Copyright by Amplyus LLC, all rights reserved Load Agarose Gel Load the gel as follows: 1.10µL DNA Ladder 2.12µL PCR product A 3.12µL PCR Product B 4.12µL PCR Product P 5.12µL PCR Product NP 6.12µL Restriction Digest AX 7.12µL Restriction Digest BX 8.12µL Restriction Digest PX 9.12µL Restriction Digest NPX

25. 24 © Copyright by Amplyus LLC, all rights reserved Questions to probe deeper – (After gel run) How did the investigation turn out? Which plant might be the source of the outbreak? Why? What’s the importance of running controls from the USDA Reference Lab? What caveats should be applied when analyzing these results? What was the most unexpected thing you learned?

26. 25 © Copyright by Amplyus LLC, all rights reserved Thank you We hope you enjoyed this lab!

27. 26 © Copyright by Amplyus LLC, all rights reserved Additional resources Outbreak Detection Since Jack in the Box: A Public Health Evolution http://www.foodsafetynews.com/2013/02/outbreak-detection-since-jack-in-the-box- a-public-health-evolution/#.VNre1J3F-Sp CDC PulseNet Home http://www.cdc.gov/pulsenet/ Centers for Disease Control: E.coli outbreaks http://www.cdc.gov/ecoli/outbreaks.html Popular books around E.coli O157:H7 outbreaks Toxin (by Robin Cook): http://en.Wikipedia.org/wiki/Toxin_%28novel%29 Poisoned (by Jeff Benedict): http://www.amazon.com/Poisoned-Deadly-Outbreak-Changed- Americans/dp/098495435X

28. 27 © Copyright by Amplyus LLC, all rights reserved Appendix: Potential variants of this lab Variant 1: Food safety inspectors & USDA Reference Lab Food Safety Inspectors: Process DNA from Plant A and Plant B USDA Reference Lab: Process control DNA (P and NP) Variant 2: Testing more than 2 plants Most lab groups receive NP DNA Selected 1-2 groups receive P DNA

29. 28 © Copyright by Amplyus LLC, all rights reserved Appendix: Copy cycles amplify DNA exponentially

30. 29 © Copyright by Amplyus LLC, all rights reserved How miniPCR TM thermal cyclers enable DNA amplification 1 3 4 1. Heated lid Prevents condensation 2 2. Heating block Separates DNA strands, preparing them for copy 3. Cooling fans Cools DNA, priming it for copy 4. Microprocessor Stores and controls temperature cycles