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Yeast Comparative Genomic Hybridization (CGH): A method for microarray detection of aneuploidy in S. cerevisiae PowerPoint Presentation
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Yeast Comparative Genomic Hybridization (CGH): A method for microarray detection of aneuploidy in S. cerevisiae

Yeast Comparative Genomic Hybridization (CGH): A method for microarray detection of aneuploidy in S. cerevisiae

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Yeast Comparative Genomic Hybridization (CGH): A method for microarray detection of aneuploidy in S. cerevisiae

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  1. Yeast Comparative Genomic Hybridization (CGH): A method for microarray detection of aneuploidy in S. cerevisiae Jackie Ryan Honors Thesis Defense April 20, 2006

  2. Overview • DNA Microarrays • Typical Use • CGH arrays • Development of CGH Procedure • Basic Method • Optimization • Validation • Future use of CGH Procedure at Davidson

  3. Typical DNA Microarray

  4. Typical DNA Microarray Process

  5. Typical DNA Microarray Process

  6. Typical DNA Microarray Process

  7. Yeast cDNA Microarray

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

  9. Laboratory Evolution • Stressed yeast = aneuploidy • Acetone & Cold, Glucose-limited conditions • Under glucose-limited conditions • Chromosomal rearrangements (Dunham et al., 2002) • Abnormal copy number of genes(Ferea et al., 1999)

  10. Human Cancers • Aneuploidy and disease • Cancer • Cell division pathways • BUB1B • Multiple hit hypothesis • Oncogenes • Tumor-suppressors

  11. Questions to Answer • Is aneuploidy random? • Is it reproducible? • Position • Sequence • Function

  12. Advantages of CGH • High-throughput • Identify: candidate genes, patterns • Compare two different populations • wild type vs. evolved • normal tissue vs. cancerous tissue

  13. 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

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

  15. Hypothetical CGH Experiment Red = wt Green = Evolved

  16. Hypothetical CGH Experiment No binding Red = wt Green = Evolved

  17. Hypothetical CGH Experiment 300: 1 Red = wt Green = Evolved

  18. Hypothetical CGH Experiment 1: 300 Red = wt Green = Evolved

  19. Hypothetical CGH Experiment 1 : 1 Red = wt Green = Evolved

  20. Genomic Isolation Method • Factors Considered • Toxicity • Time • Cost • Ease of use • Zymo kits

  21. YeaStar Genomic DNA Isolation

  22. YeaStar + ZR Genomic DNA kit >12 kb

  23. Genisphere CGH Procedure: Amount of DNA 0.3 µg ~5.0 µg

  24. Genisphere CGH Procedure: Clean DNA · Calf-thymus DNA: 18.9 ng/µL

  25. Genisphere CGH Procedure: Hybridization Buffer Buffer 5 Buffer 6 Buffer 7

  26. Genisphere CGH Procedure: Hybridization Temperature 48°C 52.5°C

  27. Genisphere CGH Procedure: Minimize Background Before After Before After

  28. Genisphere CGH Procedure Early CGH Optimized CGH Published Array

  29. Validation of CGH Procedure: Microarray wt = green ∆Sir2 = red

  30. Validation of CGH Procedure: Microarray

  31. Validation of CGH Procedure: PCR 2.0 kb 1.8 kb

  32. Validation of CGH Procedure: PCR + Nde I Digestion 2.0 kb 1.8 kb 1.8 kb 1.3 kb 0.5 kb 0.2 kb

  33. False Positives · Self vs. Self Arrays visually show consistent green and red spots

  34. False Positives · Compile numerical data - 51 spots were consistently green or red · Hypothesis: 3DNA reagent bind directly to spots · LALIGN to test

  35. False Positives

  36. Future Work with CGH Procedure • Mutant Yeast from Dr. Clifford Zeyl • Evolution under glucose-limited conditions • 2,000 generations • 5,000 generations

  37. Acknowledgments • Davidson College • Biology Department • 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