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A Comparison of Isolated Brook Trout Mitochondrial DNA from Pennsylvania

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.

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A Comparison of Isolated Brook Trout Mitochondrial DNA from Pennsylvania

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  1. 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

  2. Salvelinusfontinalis

  3. 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

  4. Impacts of AMD on Aquatic Ecosystems and Brook Trout (Salvelinusfontinalis) • Aquatic Ecosystems: • Poor water quality • Heavy metal contamination • Sedimentation • Brook Trout • Isolated populations • Uninhabitable areas due to AMD • Genetic bottlenecks • Expected genetic variation

  5. Methods of DNA Sequencing

  6. Isolation • Fin clips provided by Trout Unlimited • Preserved in 70% ethanol • GiagenGentra PUREGENE ® purification kit

  7. Courtesy: Trout Unlimited

  8. Amplification • Polymerase Chain Reaction (PCR) • Rapid amplification • D-loop region of mitochondrial DNA • 1000 base pair control region • Primers designed from published DNA sequences

  9. www.potosisd.k12.wi.us

  10. Verification • 2% Agarose Gel Electrophesis • Appearance of DNA bands verifies successful amplification • NanoDrop® Spectrophotometer ND-1000 • Determine purity/concentration of DNA

  11. Sequencing • Di-deoxy preparation • DyeEx Spin Kit purification • Sequence using ABI 310 Genetic Analyzer

  12. http://en.wikipedia.org/wiki/File:Sanger-sequencing.svg

  13. Results • Initial primers yielded 500 base pair segment • 99 to 100% homology with published genome • Lack of sufficient data to determine possible genetic variations

  14. Results Continued • Current primers yielded 900 base pair segment • Eight fish from five different locations have been sequenced • Preliminary results show minor variations

  15. Conclusions and Future Work • Additional primer sets to analyze samples • Increase sample size • Expansion of project to include nuclear DNA

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