Release v3.3.1: March 2016 © Copyright by Amplyus LLC, all rights reserved miniPCR TM Food Safety Lab Tainted Patties! Science for everyone, everywhere

1 © Copyright by Amplyus LLC, all rights reserved Welcome Our goals for today: Review key concepts in molecular microbiology Solve a public health crisis using DNA analysis Contain an outbreak of deadly foodborne illness!

2 © Copyright by Amplyus LLC, all rights reserved PCR Text PCR applications have transformed our world Forensics Food and agriculture Consumer genomics Molecular diagnostics Personalized medicine Human evolution Text

3 © Copyright by Amplyus LLC, all rights reserved Outbreak of E. coli in ground beef  Dozens of victims nationwide Ongoing USDA investigation  Investigate meat processing facilities  Test for E. coli O157:H7 strain Use of core biotechnology techniques  PCR  Restriction digest  DNA electrophoresis Real-world scenario  DNA in public health / surveillance  Biotechnology in food industry Use DNA technology to solve a real-world problem

4 © Copyright by Amplyus LLC, all rights reserved Foodborne illness crisis

5 © Copyright by Amplyus LLC, all rights reserved Foodborne pathogens: identifying the culprit bacteria Most Escherichia coli are harmless living 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, antigen detection are slow and inefficient

6 © Copyright by Amplyus LLC, all rights reserved Micropipetting Gel electrophoresis PCR Restriction digest Frist class period We will use the same tools as USDA scientists Second class period

7 © 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

8 © 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

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

10 © 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

11 © Copyright by Amplyus LLC, all rights reserved We will investigate 4 samples From Meat Packing Facility “A” Sample ASample B Field Samples Control PControl NP Non-pathogenic E.coli DNA Pathogenic E.coli DNA Controls From Meat Packing Facility “B”

12 © 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+ What goes in a PCR experiment 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

13 © 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

14 © 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

15 © Copyright by Amplyus LLC, all rights reserved Programming miniPCR ISS Food Safety Lab

16 © Copyright by Amplyus LLC, all rights reserved Foodborne Illness Outbreaks: Massive Public Health Concern 48M Americans get sick each year 128,000 are hospitalized 3,000 die Foodborne illnesses cost the economy more than $15.6 billion / yr Source: and

17 © Copyright by Amplyus LLC, all rights reserved How DNA can solve this food mystery fliC locus (non-pathogenic E.coli strain ) fliC fliC locus (pathogenic E.coli O157:H7 strain ) XmnI site Genetic difference between E.coli strains Single nucleotide polymorphism (SNP) in fliC gene SNP creates restriction site only in pathogenic E.coli O157:H7 SNP

18 © Copyright by Amplyus LLC, all rights reserved fliC Experimental plan Gel electrophoresis Non-pathogenic O157:H7 400bp bp fliC XmnI site Set-up Electrophoresis & DNA staining PCR Restriction digest 1234

19 © Copyright by Amplyus LLC, all rights reserved Monitoring DNA amplification What is happening to DNA molecules at each PCR step? Denaturation Annealing Extension What is unique about Taq DNA polymerase? What temperature is optimal for most enzymes? How many molecules of DNA will we have with each PCR cycle? And at the end of the PCR?

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

21 © Copyright by Amplyus LLC, all rights reserved Micropipetting Gel electrophoresis PCR Restriction digest First class period Second class period: Restriction Digest and Electrophoresis NOW

22 © 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) +

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

24 © Copyright by Amplyus LLC, all rights reserved Next step: gel electrophoresis to visualize PCR products 1. Pour an agarose gel2. Load the PCR products 4. Visualization in a transilluminator3. Electrophoresis e- - Pole + Pole

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26 © Copyright by Amplyus LLC, all rights reserved Load Agarose Gel Load a 2% agarose 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

27 © Copyright by Amplyus LLC, all rights reserved Questions to probe deeper – (After gel run) How did the investigation turn out? Which meat processing facility might be the source of the outbreak? 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?

28 © Copyright by Amplyus LLC, all rights reserved Thank you - We hope you enjoyed the lab! Facebook.com/miniPCR

29 © Copyright by Amplyus LLC, all rights reserved Open inquiry - variants of this lab Variant 1: Open inquiry Testing more than 2 Meat processing facilities Most lab groups receive multiple NP DNA samples Selected groups receive P DNA (~2-3 groups per class) Students must collaborate and exchange data to establish source of the outbreak Variant 2: Split roles Food safety inspectors vs. USDA Reference Lab Food Safety Inspectors: Groups process DNA Samples A and B only USDA Reference Lab: Groups process controls (P and NP) only Students must collaborate and match data to establish source of the outbreak

30 © Copyright by Amplyus LLC, all rights reserved Additional resources Outbreak Detection Since Jack in the Box: A Public Health Evolution a-public-health-evolution/#.VNre1J3F-Sp CDC PulseNet Home Centers for Disease Control: E.coli outbreaks Popular books around E.coli O157:H7 outbreaks Toxin (by Robin Cook): Poisoned (by Jeff Benedict): Americans/dp/ X

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

32 © Copyright by Amplyus LLC, all rights reserved How miniPCR TM thermal cyclers enable DNA amplification 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

33 © Copyright by Amplyus LLC, all rights reserved Real-world impact

34 © Copyright by Amplyus LLC, all rights reserved SamplesVisualizationPCRElectrophoresis The complete biotech toolkit: DNA Discovery System™