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EPA New England Regional Laboratory
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qPCR: Rapid Method Tour
Save The Harbor/Save The Bay
May 30, 2012
Scientific Excellence to Protect Public Health & the Environment
EPA New England Regional Laboratory
2 Scientific Excellence to Protect Public Health & the Environment
Overview PCR - Problem
PCR - Background & How it Works
PCR - Correlations
PCR - Present & Future
EPA New England Regional Laboratory
Scientific Excellence to Protect Public Health & the Environment 3
“Sweetheart, we won’t really know until tomorrow.”
“Mommy? When will we know if it’s safe to swim in the water?”
Problem: Bacterial culture methods yield
Results NEXT day.
Solution : Reliable rapid methods inform bathers of
Water Quality the SAME day.
EPA New England Regional Laboratory
Background Polymerase Chain Reaction (PCR) is an
analytical tool applied from the branch of science called Genomics.
Genomics is the study of organisms in terms of their DNA sequences and cellular activity in response to the environment.
4 Scientific Excellence to Protect Public Health & the Environment
EPA New England Regional Laboratory
Scientific Excellence to Protect Public Health & the Environment 5
Background
EPA New England Regional Laboratory
Dr. Kary Mullis received a Nobel Prize in chemistry in 1993 for his invention of the polymerase chain reaction (PCR).
Conceptualized in 1983 by Dr. Mullis, The process is hailed as one of the monumental scientific techniques of the 20th century.
Scientific Excellence to Protect Public Health & the Environment 6
Background -The Inventor
EPA New England Regional Laboratory
Scientific Excellence to Protect Public Health & the Environment 7
Genomics is the science of gene…
Identification
Structure, and
Function
How It Works
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Scientific Excellence to Protect Public Health & the Environment 8
PCR is … • AKA: Repetitive Molecular Photocopying
• Utilizes Polymerase: natural enzyme catalyst DNA replication
• Analytical method that mimics the process of cellular DNA duplication
• Method Premise - Many copies are easier to identify than fewer copies
• TV Crime Shows: NCIS, CSI-Miami, etc.
EPA New England Regional Laboratory
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to generate millions - billions of copies of target gene sequences through repetitive cycling of the 3 PCR steps
Polymerase Chain Reaction (PCR):
The goal of PCR is…
- uses the natural functions of the enzyme polymerase - to copy, genetic material (e.g., DNA or RNA)
- in a simple 3 step process:
1. Denaturation 2. Annealing 3. Extension
How It Works
EPA New England Regional Laboratory
Scientific Excellence to Protect Public Health & the Environment 10
WHY AMPLIFY DNA?
In traditional microbiology bacterial cells are grown or “amplified” to form colonies which can be easily identified and counted.
In PCR sections of DNA , unique target gene sequences, are copied or amplified instead of whole cells.
Target
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How It Works Target Gene
≈ 2n copies per cycle
1st cycle
2nd cycle
3rd cycle
4th cycle
From 1 original copy
35 - 45 PCR Cycles ≈ 40 - 60 minutes
235 = 34 Billion
Copies To Millions or Billions of Copies
EPA New England Regional Laboratory
Scientific Excellence to Protect Public Health & the Environment 12
Detection & Concentration
EPA New England Regional Laboratory
Scientific Excellence to Protect Public Health & the Environment 13
A Word About Correlations
• Be Careful • Know Why • qPCR & cult. results corr. are…
• Site specific • Temporally specific
• qPCR & epidemiology corr. are… • More important
• Show higher corr. than
EPA New England Regional Laboratory
Scientific Excellence to Protect Public Health & the Environment 14
1 10
100 1,000
10,000 100,000
1,000,000 10,000,000
Site
Ente
roco
ccus
/ 10
0mL
PCR CCE PCR CFU Enterolert MPN
Enterococci Concentration
EPA New England Regional Laboratory
qPCR & Epi Data
Scientific Excellence to Protect Public Health & the Environment 15
EPA New England Regional Laboratory
2012 Published new EPA qPCR Recreational Water
Method with DNA-based Rec Water Criteria Source Tracking Apps & Research cont’d
2012 eGST RARE Human Vs. Nonhuman
Fecal Indicator Organisms (FIOs)
Tool Box
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2013 qPCR Method Training
EPA New England Regional Laboratory
References & Links EPA Genomics Task Force White Paper Inside PCR
http://learn.genetics.utah.edu/code/cfpages/cite.cfm?title=PCR%20Virtual%20Lab
Kary Mullis:
http://www.karymullis.com/index.shtml
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EPA New England Regional Laboratory
Questions
Scientific Excellence to Protect Public Health & the Environment 18
EPA New England Regional Laboratory
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PCR Sample to Results
EPA New England Regional Laboratory
PCR is a powerful analytical tool for supporting a broad spectrum of EPA Programs:
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Superfund: Risk Assessment
(e.g., Estrogen, Hg, etc.)
Bioremediation
NPDES: Toxicity Testing
TSCA:
Nanotechnology
TMDL: Microbial Source Tracking
Source Identification
CAFOs Drinking Water:
Rapid Screening
BEACHES:
Rapid Water Testing
Scientific Excellence to Protect Public Health & the Environment
How We Use It
EPA New England Regional Laboratory
Scientific Excellence to Protect Public Health & the Environment 21
Robustness
EPA New England Regional Laboratory
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Specificity
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