PCR Amplification of

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PCR Amplification of Environmental DNA
tutorial
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Applications
1. Identify microorganisms 2. Quantify
microorganisms 3. Assess bioremediation 4.
Research ecology, evolution, paleontology
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An Ecological Example
Environ. Sci. Technol., 40 (23), 7445 -7450,
2006. 10.1021/es060413l S0013-936X(06)00413-5 Ant
ibiotic Resistance Genes as Emerging
Contaminants Studies in Northern Colorado Amy
Pruden, Ruoting Pei, Heather Storteboom, and
Kenneth H. Carlson
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ScienceDaily - October 26, 2006
Antibiotic resistance genes (ARGs) should be
considered emerging environmental contaminants
with more research devoted to the mechanisms by
which they spread, scientists say in a report
scheduled for the Dec. 1 issue of the
semi-monthly ACS journal Environmental Science
Technology. Colorado State University's Amy
Pruden and colleagues reached that conclusion
after a study that documented occurrence of
tetracycline and sulfonamide ARGs in irrigation
ditches, river sediments, and other spots in the
environment in northern Colorado. ARGs are
pieces of DNA that make bacteria resistant to
common antibiotics - recognized as an
increasingly serious global health problem. The
genes can spread in different ways. Bacteria, for
instance, exchange ARGs among themselves. Pruden
and colleagues note that even if cells carrying
ARGs have been killed, DNA released to the
environment can persist and spread to other
cells. "ARGs in and of themselves can be
considered to be emerging 'contaminants' for
which mitigation strategies are needed to prevent
their widespread dissemination," they state.
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Environmental PCR
Cons
Pros
Save time More robust More information
Controls Startup cost Training
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Challenge
Extracting DNA from soil samples clean enough for
PCR is difficult. Compounds exist in soil that
inhibit PCR. Can we isolate enough DNA for PCR
on a high school budget?
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Bacterial Genomic DNA?
Soil supernatant
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Design
1. Supernatant 2. Supernatant 1/10 dilution 3.
Precipitate from supernatant 4. Precipitate from
supernatant 1/10 dilution 5. Gel-purification 6.
Gel-purification precipitate 7. Streaked
supernatant
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Target
Primers chosen to amplify 16S ribosomal RNA
subunit common in all bacteria. Positive PCR
result indicates presence of bacteria.
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Controls
1. Water only (- control) 2. Water primers (-
control) 3. E. coli colony ( control)
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PCR Programs
Many thanks to Amy Pruden and Luciana Pereyra _at_
Colorado State University
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Future Direction
To develop a high school-friendly PCR protocol
for students to use to identify specific
microbes. Students will have to use
bioinformatic techniques to select robust primers
for their organism of interest.