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

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 0.5 mL yeast cells 1.5 mL yeast cells Concentration (ng/ µ L) 31.743.1 Absorption at 260 nm 0.6340.861 Ratio 260/280 1.751.86

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

14. Genisphere CGH Procedure Amount of DNA Clean Tagged DNA Hybridization Buffer Hybridization Temperature Minimize Background BeforeAfter

15. False Positives False Positive RedsAverage Red/Green RatioRed 5'-3': Identity Score (Upper Limit =44) YLR243W32981 YIL012W12.863 YKL215C5.559 YOR296W23.649 False Positive GreensAverage Green/Red RatioGreen 5'-3': Identity Score (Upper Limit =49) YEL020W-A5555.578 YBR162W-A5524.966 YCR094W393.560 YGR153W2652.560 YBR006WOff scale58 YLR174W138.853 YHR057C191.753 YLR087COff scale51 YBR034COff scale51 YNL015W277.550 YOR046COff scale50

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