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

1
Yeast Comparative Genomic Hybridization (CGH)
A method for microarray detection of aneuploidy
in S. cerevisiae

• Jackie Ryan
• Honors Thesis Defense
• April 20, 2006

2
Overview

• DNA Microarrays
• Typical Use
• CGH arrays
• Development of CGH Procedure
• Basic Method
• Optimization
• Validation
• Future use of CGH Procedure at Davidson

3
Typical DNA Microarray
4
Typical DNA Microarray Process
5
Typical DNA Microarray Process
6
Typical DNA Microarray Process
7
Yeast cDNA Microarray
8
CGH An Alternative Use of DNA Microarrays

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

9
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)

10
Human Cancers

• Aneuploidy and disease
• Cancer
• Cell division pathways
• BUB1B
• Multiple hit hypothesis
• Oncogenes
• Tumor-suppressors

11
Questions to Answer

• Is aneuploidy random?
• Is it reproducible?
• Position
• Sequence
• Function

12
Advantages of CGH

• High-throughput
• Identify candidate genes, patterns
• Compare two different populations
• wild type vs. evolved
• normal tissue vs. cancerous tissue

13
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

14
Tagging Method

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

15
Hypothetical CGH Experiment
Red wt Green Evolved
16
Hypothetical CGH Experiment
No binding
Red wt Green Evolved
17
Hypothetical CGH Experiment
300 1
Red wt Green Evolved
18
Hypothetical CGH Experiment
1 300
Red wt Green Evolved
19
Hypothetical CGH Experiment
1 1
Red wt Green Evolved
20
Genomic Isolation Method

• Factors Considered
• Toxicity
• Time
• Cost
• Ease of use
• Zymo kits

21
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
22
YeaStar ZR Genomic DNA kit
wt S288C Evolved L?42K
Concentration (ng/µL) 122 35.2
Absorption at 260 nm 0.245 0.703
Ratio 260/280 1.91 1.90
gt12 kb
23
Genisphere CGH Procedure Amount of DNA
0.3 µg
5.0 µg
24
Genisphere CGH Procedure Clean DNA
Calf-thymus DNA 18.9 ng/µL
Zymo kit Promega kit
Percent yield 90.6 74.9
25
Genisphere CGH Procedure Hybridization Buffer
Buffer 5
Buffer 6
Buffer 7
26
Genisphere CGH Procedure Hybridization
Temperature
48C
52.5C
27
Genisphere CGH Procedure Minimize Background
Before
After
Before
After
28
Genisphere CGH Procedure
Early CGH
Optimized CGH
Published Array
29
Validation of CGH Procedure Microarray
wt green ?Sir2 red
30
Validation of CGH Procedure Microarray
Gene Name Avg. Ratio (?Sir2/wt) Standard Deviation Chromosome Biological Process Molecular Function Cellular Component
YAR020C 0.482 0.371 1 unknown unknown unknown
YBR123C 0.503 0.420 2 Transcription initiation Pol III promoter RNA poly. III transcription factor Transcription factor TFIIIC complex
YBR169C 0.355 0.358 2 Protein folding Heat shock protein activity unknown
YDL042C 0.502 0.420 4 Chromatin silencing at telomere Histone deacetylase activity nucleolus
YDR087C 0.480 0.357 4 rRNA processing unknown unknown
YDR131C 0.548 0.371 4 Ubiquitin-dependent protein catabolism Protein binding activity Ubiquitin ligase complex
YDR211W 0.490 0.204 4 Translation initiation Translation initiation factor activity Ribosome
YER102W 0.383 0.245 5 Protein biosynthesis Structural constituent of ribosome Cytosolic small ribosomal subunit (sensu Eukarya)
YER104W 0.439 0.390 5 Neg. reg. of DNA transposition unknown unknown
YGR072W 0.441 0.217 7 mRNA catabolism unknown Cytoplasm
YOL162W 0.546 0.440 15 Transport Transporter activity Membrane
YPR049C 0.500 0.404 16 Protein-vacuolar targeting unknown Extrinsic to membrane
31
Validation of CGH Procedure PCR
2.0 kb
1.8 kb
32
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
33
False Positives
Self vs. Self Arrays visually show consistent
green and red spots
34
False Positives
Compile numerical data - 51 spots were
consistently green or red Hypothesis 3DNA
reagent bind directly to spots LALIGN to test
35
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
36
Future Work with CGH Procedure

• Mutant Yeast from Dr. Clifford Zeyl
• Evolution under glucose-limited conditions
• 2,000 generations
• 5,000 generations

37
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