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A. Jacqueline Ryan Davidson College March 25, 2006

Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae. A. Jacqueline Ryan Davidson College March 25, 2006. Overview. DNA Microarrays Development of CGH Procedure Basic Method Optimization Validation

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A. Jacqueline Ryan Davidson College March 25, 2006

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  1. Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson College March 25, 2006

  2. Overview • DNA Microarrays • Development of CGH Procedure • Basic Method • Optimization • Validation • Applications of CGH Procedure

  3. Traditional DNA Microarray Process

  4. Traditional DNA Microarray Process

  5. Traditional DNA Microarray Process

  6. Traditional DNA Microarray Process

  7. Yeast Microarray

  8. CGH: An Alternative Use of DNA Microarrays • Hybridize genomic DNA to array • Detect deletions/amplifications of genes (aneuploidy) • Applications: • Evolution • Cancer

  9. Outline of CGH Process 1. Isolate Genomic DNA 2. Fragment DNA 3. Tag DNA 4. Hybridize Tagged DNA 5. Hybridize 3DNA reagent 6. Scan Array 7. Analyze data

  10. Tagging Method • Genisphere 3DNA Array 900DNA kit Alexa 546/Alexa 647 • Robust Signal • Less photobleaching

  11. Genomic Isolation Method • Factors Considered • Toxicity • Time • Cost • Ease of use • Zymo YeaStar Genomic DNA kit

  12. YeaStar Genomic DNA Isolation

  13. Genisphere CGH Procedure Early CGH • Amount of DNA • Clean Tagged DNA • Hybridization Buffer • Hybridization Temperature • Minimize Background Optimized CGH

  14. Genisphere CGH Procedure Before After • Amount of DNA • Clean Tagged DNA • Hybridization Buffer • Hybridization Temperature • Minimize Background

  15. False Positives

  16. Validation of CGH Procedure

  17. Future Work with CGH Procedure • Mutant Yeast from Dr. Clifford Zeyl • Evolution under glucose limiting conditions • 2000 generations • 5000 generations

  18. Acknowledgments • Davidson College • Dr. Malcolm Campbell • Dr. Laurie Heyer • Dr. Karen Bernd • Chris Healey • Peggy Maiorano • Emily Oldham and previous genomics students • Lab mates: Matt Gemberling, Mac Cowell, Kristen DeCelle, Franois Trappey, Andrew Drysdale, and Oscar Hernandez • Other Institutions • Dr. Todd Eckdahl of Missouri Western State College • Dr. Laura Hoopes of Pomona College • Dr. Clifford Zeyl of Wake Forest University • GCAT • Genisphere and Zymo Research

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