a comparison of isolated brook trout mitochondrial dna from pennsylvania
DESCRIPTION
A Comparison of Isolated Brook Trout Mitochondrial DNA from Pennsylvania. Fred J. Brenner, Ph.D.* Shawn M. Rummel, Ph.D ** Nicholas J. Petersen*,Christina R. Cropsey *, Lauren M. McGarvey*, *Grove City College, **Trout Unlimited, Eastern Abandoned Mine Program. Salvelinus fontinalis. - PowerPoint PPT PresentationTRANSCRIPT
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A Comparison of Isolated Brook Trout Mitochondrial DNA from Pennsylvania
Fred J. Brenner, Ph.D.*Shawn M. Rummel, Ph.D**Nicholas J. Petersen*,Christina R. Cropsey*, Lauren M. McGarvey*,
*Grove City College, **Trout Unlimited, Eastern Abandoned Mine Program
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Salvelinus fontinalis
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Introduction •Coal mining and abandoned mine drainage
▫Pyrite exposed in abandoned mines▫Oxidation to sulfuric acid ▫Ground water contamination
http://www.tu.org/conservation/abandoned-mines/amd-101
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Impacts of AMD on Aquatic Ecosystems and Brook Trout (Salvelinus fontinalis)•Aquatic Ecosystems:
▫Poor water quality▫Heavy metal contamination ▫Sedimentation
•Brook Trout▫Isolated populations ▫Uninhabitable areas due to AMD▫Genetic bottlenecks ▫Expected genetic variation
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Methods of DNA Sequencing
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Isolation •Fin clips provided by Trout Unlimited •Preserved in 70% ethanol •Giagen Gentra PUREGENE ® purification
kit
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Courtesy: Trout Unlimited
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Amplification •Polymerase Chain Reaction (PCR)
▫Rapid amplification •D-loop region of mitochondrial DNA
▫1000 base pair control region •Primers designed from published DNA
sequences
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www.potosisd.k12.wi.us
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Verification •2% Agarose Gel Electrophesis
▫Appearance of DNA bands verifies successful amplification
•NanoDrop ® Spectrophotometer ND-1000 ▫Determine purity/concentration of DNA
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Sequencing •Di-deoxy preparation•DyeEx Spin Kit purification •Sequence using ABI 310 Genetic Analyzer
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http://en.wikipedia.org/wiki/File:Sanger-sequencing.svg
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Results • Initial primers yielded 500 base pair
segment • 99 to 100% homology with published
genome • Lack of sufficient data to determine
possible genetic variations
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Results Continued •Current primers yielded 900 base pair
segment•Eight fish from five different locations
have been sequenced•Preliminary results show minor variations
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Conclusions and Future Work •Additional primer sets to analyze samples•Increase sample size•Expansion of project to include nuclear
DNA