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Yeast Comparative Genomic Hybridization (CGH): A method for microarray detection of aneuploidy in S. cerevisiae. Jackie Ryan Honors Thesis Defense April 20, 2006. Overview. DNA Microarrays Typical Use CGH arrays Development of CGH Procedure Basic Method Optimization Validation
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Yeast Comparative Genomic Hybridization (CGH): A method for microarray detection of aneuploidy in S. cerevisiae Jackie Ryan Honors Thesis Defense April 20, 2006
Overview • DNA Microarrays • Typical Use • CGH arrays • Development of CGH Procedure • Basic Method • Optimization • Validation • Future use of CGH Procedure at Davidson
CGH: An Alternative Use of DNA Microarrays • Hybridize genomic DNA to array • Detect deletions/amplifications of genes (aneuploidy) • Applications: • Laboratory Evolution • Cancer
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)
Human Cancers • Aneuploidy and disease • Cancer • Cell division pathways • BUB1B • Multiple hit hypothesis • Oncogenes • Tumor-suppressors
Questions to Answer • Is aneuploidy random? • Is it reproducible? • Position • Sequence • Function
Advantages of CGH • High-throughput • Identify: candidate genes, patterns • Compare two different populations • wild type vs. evolved • normal tissue vs. cancerous tissue
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
Hypothetical CGH Experiment Red = wt Green = Evolved
Hypothetical CGH Experiment No binding Red = wt Green = Evolved
Hypothetical CGH Experiment 300: 1 Red = wt Green = Evolved
Hypothetical CGH Experiment 1: 300 Red = wt Green = Evolved
Hypothetical CGH Experiment 1 : 1 Red = wt Green = Evolved
Genomic Isolation Method • Factors Considered • Toxicity • Time • Cost • Ease of use • Zymo kits
YeaStar + ZR Genomic DNA kit >12 kb
Genisphere CGH Procedure: Amount of DNA 0.3 µg ~5.0 µg
Genisphere CGH Procedure: Clean DNA · Calf-thymus DNA: 18.9 ng/µL
Genisphere CGH Procedure: Hybridization Buffer Buffer 5 Buffer 6 Buffer 7
Genisphere CGH Procedure: Hybridization Temperature 48°C 52.5°C
Genisphere CGH Procedure: Minimize Background Before After Before After
Genisphere CGH Procedure Early CGH Optimized CGH Published Array
Validation of CGH Procedure: Microarray wt = green ∆Sir2 = red
Validation of CGH Procedure: PCR 2.0 kb 1.8 kb
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
False Positives · Self vs. Self Arrays visually show consistent green and red spots
False Positives · Compile numerical data - 51 spots were consistently green or red · Hypothesis: 3DNA reagent bind directly to spots · LALIGN to test
Future Work with CGH Procedure • Mutant Yeast from Dr. Clifford Zeyl • Evolution under glucose-limited conditions • 2,000 generations • 5,000 generations
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