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 Applications of CGH Procedure

Traditional DNA Microarray Process

Yeast Microarray

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

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

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

Genomic Isolation Method Factors Considered –Toxicity –Time –Cost –Ease of use Zymo YeaStar Genomic DNA kit

YeaStar Genomic DNA Isolation 0.5 mL yeast cells 1.5 mL yeast cells Concentration (ng/ µ L) Absorption at 260 nm Ratio 260/

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

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

False Positives False Positive RedsAverage Red/Green RatioRed 5'-3': Identity Score (Upper Limit =44) YLR243W32981 YIL012W YKL215C5.559 YOR296W False Positive GreensAverage Green/Red RatioGreen 5'-3': Identity Score (Upper Limit =49) YEL020W-A YBR162W-A YCR094W YGR153W YBR006WOff scale58 YLR174W YHR057C YLR087COff scale51 YBR034COff scale51 YNL015W YOR046COff scale50

Validation of CGH Procedure

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

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