yeast comparative genomic hybridization (cgh): a streamlined method for microarray detection of...
TRANSCRIPT
![Page 1: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/1.jpg)
Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae
A. Jacqueline Ryan
Davidson College
March 25, 2006
![Page 2: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/2.jpg)
Overview
• DNA Microarrays
• Development of CGH Procedure– Basic Method– Optimization– Validation
• Applications of CGH Procedure
![Page 3: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/3.jpg)
Traditional DNA Microarray Process
![Page 4: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/4.jpg)
Traditional DNA Microarray Process
![Page 5: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/5.jpg)
Traditional DNA Microarray Process
![Page 6: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/6.jpg)
Traditional DNA Microarray Process
![Page 7: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/7.jpg)
Yeast Microarray
![Page 8: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/8.jpg)
CGH: An Alternative Use of DNA Microarrays
• Hybridize genomic DNA to array
• Detect deletions/amplifications of genes (aneuploidy)
• Applications:– Evolution– Cancer
![Page 9: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/9.jpg)
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
![Page 10: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/10.jpg)
Tagging Method
• Genisphere 3DNA Array 900DNA kit Alexa 546/Alexa 647– Robust Signal– Less photobleaching
![Page 11: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/11.jpg)
Genomic Isolation Method
• Factors Considered– Toxicity– Time– Cost– Ease of use
• Zymo YeaStar Genomic DNA kit
![Page 12: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/12.jpg)
YeaStar Genomic DNA Isolation
0.5 mL yeast cells
1.5 mL yeast cells
Concentration (ng/µL) 31.7 43.1
Absorption at 260 nm 0.634 0.861
Ratio 260/280 1.75 1.86
![Page 13: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/13.jpg)
Genisphere CGH Procedure
• Amount of DNA
• Clean Tagged DNA
• Hybridization Buffer
• Hybridization Temperature
• Minimize Background
Early CGH
Optimized CGH
![Page 14: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/14.jpg)
Genisphere CGH Procedure
• Amount of DNA
• Clean Tagged DNA
• Hybridization Buffer
• Hybridization Temperature
• Minimize Background
Before After
![Page 15: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/15.jpg)
False PositivesFalse Positive Reds Average Red/Green Ratio Red 5'-3': Identity Score
(Upper Limit =44)
YLR243W 329 81
YIL012W 12.8 63
YKL215C 5.5 59
YOR296W 23.6 49
False Positive Greens Average Green/Red Ratio Green 5'-3': Identity Score (Upper Limit =49)
YEL020W-A 5555.5 78
YBR162W-A 5524.9 66
YCR094W 393.5 60
YGR153W 2652.5 60
YBR006W Off scale 58
YLR174W 138.8 53
YHR057C 191.7 53
YLR087C Off scale 51
YBR034C Off scale 51
YNL015W 277.5 50
YOR046C Off scale 50
![Page 16: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/16.jpg)
Validation of CGH Procedure
![Page 17: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/17.jpg)
Future Work with CGH Procedure
• Mutant Yeast from Dr. Clifford Zeyl– Evolution under glucose limiting conditions
• 2000 generations
• 5000 generations
![Page 18: Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae A. Jacqueline Ryan Davidson](https://reader033.vdocuments.us/reader033/viewer/2022061604/5697bff21a28abf838cbbaeb/html5/thumbnails/18.jpg)
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