Yeast Comparative Genomic Hybridization (CGH): A streamlined method for microarray detection of aneuploidy in S. cerevisiae

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Yeast Comparative Genomic Hybridization (CGH) A
streamlined method for microarray detection of
aneuploidy in S. cerevisiae

• A. Jacqueline Ryan
• Davidson College
• March 25, 2006

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Overview

• DNA Microarrays
• Development of CGH Procedure
• Basic Method
• Optimization
• Validation
• Applications of CGH Procedure

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Traditional DNA Microarray Process
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Traditional DNA Microarray Process
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Traditional DNA Microarray Process
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Traditional DNA Microarray Process
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Yeast Microarray
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CGH An Alternative Use of DNA Microarrays

• Hybridize genomic DNA to array
• Detect deletions/amplifications of genes
  (aneuploidy)
• Applications
• Evolution
• Cancer

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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

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Tagging Method

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

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Genomic Isolation Method

• Factors Considered
• Toxicity
• Time
• Cost
• Ease of use
• Zymo YeaStar Genomic DNA kit

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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
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Genisphere CGH Procedure
Early CGH

• Amount of DNA
• Clean Tagged DNA
• Hybridization Buffer
• Hybridization Temperature
• Minimize Background

Optimized CGH
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Genisphere CGH Procedure
Before
After

• Amount of DNA
• Clean Tagged DNA
• Hybridization Buffer
• Hybridization Temperature
• Minimize Background

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False Positives
False 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
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Validation of CGH Procedure
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Future Work with CGH Procedure

• Mutant Yeast from Dr. Clifford Zeyl
• Evolution under glucose limiting conditions
• 2000 generations
• 5000 generations

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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