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

Traditional DNA Microarray Process

Traditional DNA Microarray Process

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/µL) 31.7 43.1

Absorption at 260 nm 0.634 0.861

Ratio 260/280 1.75 1.86

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

Before After

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

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