Science for everyone, everywhere
DNA amplification and analysis:
TM
miniPCR Food Safety Lab
Release date: November 2015
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Welcome
Our goals for today:
• Review DNA amplification theory
• Solve a public health problem using DNA analysis
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1
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
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2
PCR relies on DNA’s unique structure
DNA: a double helix...
...held together by base complementarity
Source: US National Library of Medicine, NIH, Thinkquest
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3
How PCR works: 3 steps to copy DNA
94°C
1
Denaturation
2
50-60°C
Primer 1
Annealing
Primer 2
72°C
3
Extension
Taq DNA
polymerase
dNTPs
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4
How PCR works: repeat the cycle
denatured DNA
Single molecule
DNA + primers
DNA + copy
94° C
~1B copies
72° C
50-60° C
Denaturation
Annealing
Extension
Repeat x ~25-30 cycles
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5
PCR makes DNA visible (and useful)
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6
PCR is central to biomedical applications
Molecular
diagnostics
Text
Consumer
genomics
Personalized
medicine
Text
Food and
agriculture
Text
PCR
Text
Text
Human
evolution
Text
Forensics
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7
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
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8
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
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9
Outbreaks are a serious public health concern
Source: http://www.cdc.gov/ecoli/general/index.html#what_shiga and
http://www.cdc.gov/ecoli/outbreaks.html
•
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
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In the news
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11
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
fliC
(non-pathogenic)
XmnI site
fliC locus
(pathogenic O157:H7)
fliC
SNP
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12
Lab plan
Template
DNA
PCR
amplification
Restriction
digest
400bp
400bp
400bp
150+250bp
Gel
electrophoresis
fliC
Meat plant A
non-pathogenic
XmnI site
fliC
Meat plant B
O157:H7
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13
What goes in a PCR reaction
1. Template DNA to be amplified
2. Pair of DNA primers
Taq
3. DNA polymerase
FWD primer
REV primer
4. dNTPs
G
5. Buffer to maintain pH and provide Mg2+
A
A
C TG
G
TA
C
C
6. Thermal cycler
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14
Setting up your PCR reactions
Label 4 PCR tubes per group
Tube “A”
FIELD
SAMPLES
A.
B.
DNA sample
from meat packing
plant “A”
Tube “B”
B.
Tube “P”
USDA
“REFERENCE
LAB”
H.
I.
Control DNA
from pathogenic
E.coli
DNA sample
from meat packing
plant “B”
Tube “NP”
D.
Control DNA
from non-pathogenic
E.coli
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15
Prepare 4 PCR tubes (200µL tubes)
2X EZ PCR Master mix
15 µL
(EZ PCR Master Mix: PCR Buffer + Mg2+ + Taq + dNTPs)
3X Food Safety Primer mix
10 µL
5 µL
(Forward and Reverse primers)
DNA sample
A
DNA sample
B
Control P
DNA
Control NP
DNA
A
B
P
NP
Also add your initials to side of tube
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16
Programming PCR parameters
Initial denaturation:
94°C
30 seconds
Denaturation:
Annealing
Extension
94°C
57°C
72°C
5 seconds
5 seconds
5 seconds
x25 cycles (if prioritizing speed) OR
x30 cycles (if prioritizing robustness of results)
Final extension
72°C
30 seconds
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17
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?
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18
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
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19
Set up restriction digest (XmnI)
PCR
product
A
15 µL
+
1 µL
Restriction
digest
(incubate at
37°C)
B
P
NP
Restriction enzyme
(XmnI restriction endonuclease)
AX
BX
PX
NPX
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20
Incubate 10 minutes at 37°C
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21
Next step: visualize the DNA fragments amplified by PCR
1. Pour an agarose gel
3. Electrophoresis
2. Load the PCR products
4. Visualization in a transilluminator
- Pole
e+ Pole
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22
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
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23
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?
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24
Thank you
We hope you enjoyed this lab!
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25
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-boxa-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-ChangedAmericans/dp/098495435X
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26
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
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27
Appendix: Copy cycles amplify DNA exponentially
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28
How miniPCRTM thermal cyclers enable DNA amplification
1. Heated lid
• Prevents condensation
1
2. Heating block
• Separates DNA strands,
preparing them for copy
2
3. Cooling fans
• Cools DNA, priming it
for copy
3
4
4. Microprocessor
• Stores and controls
temperature cycles
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