Yeast Comparative Genomic Hybridization CGH A streamlined method

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

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

Genomic Isolation Method • Factors Considered – – Toxicity Time Cost Ease of use • Zymo Yea. Star Genomic DNA kit

Yea. Star Genomic DNA Isolation 0. 5 m. L yeast cells 1. 5 m. L yeast cells Concentration (ng/µL) 31. 7 43. 1 Absorption at 260 nm 0. 634 0. 861 Ratio 260/280 1. 75 1. 86

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

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

False Positives False Positive Reds Average Red/Green Ratio Red 5'-3': Identity Score (Upper Limit =44) YLR 243 W 329 81 YIL 012 W 12. 8 63 YKL 215 C 5. 5 59 YOR 296 W 23. 6 49 False Positive Greens Average Green/Red Ratio Green 5'-3': Identity Score (Upper Limit =49) YEL 020 W-A 5555. 5 78 YBR 162 W-A 5524. 9 66 YCR 094 W 393. 5 60 YGR 153 W 2652. 5 60 YBR 006 W Off scale 58 YLR 174 W 138. 8 53 YHR 057 C 191. 7 53 YLR 087 C Off scale 51 YBR 034 C Off scale 51 YNL 015 W 277. 5 50 YOR 046 C Off scale 50

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 De. Celle, 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