DNA amplification and analysis: minipcr TM Food Safety Lab

Transcription

1 Science for everyone, everywhere DNA amplification and analysis: minipcr TM Food Safety Lab Release date: 09 September 2014

2 Welcome Our goals for today: Review DNA amplification theory Solve a public health problem using DNA analysis 1

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

4 PCR relies on DNA s unique structure DNA: a double helix......held together by base complementarity Source: US National Library of Medicine, NIH, Thinkquest 3

5 How PCR works: 3 steps to copy DNA 1 94 C Denaturation 2 Primer C Annealing Primer 2 3 Extension Taq DNA polymerase dntps 72 C 4

6 How PCR works: repeat the cycle denatured DNA Single molecule DNA + primers DNA + copy 94 C ~1B copies 72 C C Denaturation Annealing Extension Repeat x ~25-30 cycles 5

7 PCR makes DNA visible (and useful) 6

8 DNA amplification is central to biomedical technology Molecular diagnostics Text Consumer genomics Personalized medicine Text Text Food and agriculture Text PCR Text Human evolution Text Forensics 7

9 We will use DNA technology to solve a real-world problem 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 8

10 Scientists work hard at detecting E. coli 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 9

11 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 Source: and Why is PCR useful? PCR is ideal for fast detection of low concentrations of organisms e.g., 15 cells per g 10

12 How biotechnology can help 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 flic locus (non-pathogenic) flic XmnI site flic locus (pathogenic O157:H7) flic SNP 11

13 Lab plan Template DNA PCR amplification Restriction digest Gel electrophoresis flic 400bp 400bp Meat plant A non-pathogenic XmnI site 400bp bp flic Meat plant B O157:H7 12

14 What goes in a PCR reaction 1. Template DNA to be amplified 2. Pair of DNA primers 3. DNA polymerase FWD primer Taq REV primer 4. dntps 5. Buffer to maintain ph and provide Mg 2+ A G A C TG G T A C C 6. Thermal cycler 13

15 Setting up your PCR reactions Label 4 PCR tubes per group Tube A Tube B FIELD SAMPLES A. Burger DNA sample from meat plant A B. Burger DNA sample from meat plant B USDA REFERENCE LAB Tube P H. Control DNA from pathogenic I. E.coli Tube NP D. Control DNA from non-pathogenic E.coli 14

16 Let s pipette 1. PCR Mix 15 µl per tube Same in all tubes H 2 0 Buffer DNA polymerase dntps 2. Primers 10 µl per tube Same primer in all tubes 3. DNA 5 µl per tube Tube A: Meat Plant A DNA sample Tube B: Meat Plant B DNA sample Tube P: Control DNA (pathogenic) Tube NP: Control DNA (non-pathogenic) 30 µl per tube A B 15

17 Programming PCR parameters Initial denaturation: 94 C 30 seconds Denaturation: 94 C 5 seconds Annealing 57 C 5 seconds Extension 72 C 5 seconds x25 cycles (if prioritizing speed) OR x30 cycles (if prioritizing robustness of results) Final extension 72 C 30 seconds 16

18 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? 17

19 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 18

20 Add restriction enzyme mix PCR product Tube A+X: Meat Plant A DNA sample Tube B:+X Meat Plant B DNA sample Tube P+X: Control DNA (pathogenic) Tube NP+X: Control DNA (non-pathogenic) 15 µl per tube Restriction enzyme mix Same in all tubes Add 5 µl per tube Incubate 15 minutes at 37 C 19

21 Next step: visualize the DNA fragments amplified by PCR 1. Pour an agarose gel 2. Load the PCR products 3. Electrophoresis 4. Visualization in a transilluminator - Pole e- + Pole 20

22 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? 21

23 Thank you We hope you enjoyed this lab! 22

24 Additional resources DNA Learning Center PCR animations University of Utah PCR Virtual Lab 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 23

25 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 24

26 Appendix: Copy cycles amplify DNA exponentially 25

27 How minipcr TM thermal cyclers enable DNA amplification 1. Heated lid Prevents condensation 2. Heating block Separates DNA strands, preparing them for copy 3. Cooling fans Cools DNA, priming it for copy Microprocessor Stores and controls temperature cycles 26