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

Typical DNA Microarray

Typical DNA Microarray Process

Yeast cDNA Microarray

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 Red = wt Green = Evolved No binding

Hypothetical CGH Experiment Red = wt Green = Evolved 300: 1

Hypothetical CGH Experiment Red = wt Green = Evolved 1: 300

Hypothetical CGH Experiment Red = wt Green = Evolved 1 : 1

Genomic Isolation Method Factors Considered –Toxicity –Time –Cost –Ease of use Zymo kits

YeaStar Genomic DNA Isolation 0.5 mL yeast cells1.5 mL yeast cells Concentration (ng/ µ L) Absorption at 260 nm Ratio 260/

YeaStar + ZR Genomic DNA kit wt S288C Evolved L  42K Concentration (ng/ µ L) Absorption at 260 nm Ratio 260/ >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 Zymo kitPromega kit Percent yield 90.6% 74.9%

Genisphere CGH Procedure: Hybridization Buffer Buffer 5 Buffer 6Buffer 7

Genisphere CGH Procedure: Hybridization Temperature 48°C52.5°C

Genisphere CGH Procedure: Minimize Background Before AfterBeforeAfter

Genisphere CGH Procedure Early CGH Optimized CGH Published Array

Validation of CGH Procedure: Microarray wt = green ∆Sir2 = red

Validation of CGH Procedure: Microarray Gene Name Avg. Ratio (∆Sir2/wt) Standard Deviation ChromosomeBiological Process Molecular Function Cellular Component YAR020C unknown YBR123C Transcription initiation Pol III promoter RNA poly. III transcription factor Transcription factor TFIIIC complex YBR169C Protein foldingHeat shock protein activityunknown YDL042C Chromatin silencing at telomere Histone deacetylase activity nucleolus YDR087C rRNA processingunknown YDR131C Ubiquitin-dependent protein catabolism Protein binding activityUbiquitin ligase complex YDR211W Translation initiationTranslation initiation factor activity Ribosome YER102W Protein biosynthesisStructural constituent of ribosome Cytosolic small ribosomal subunit (sensu Eukarya) YER104W Neg. reg. of DNA transposition unknown YGR072W mRNA catabolismunknownCytoplasm YOL162W TransportTransporter activityMembrane YPR049C Protein-vacuolar targetingunknownExtrinsic to membrane

Validation of CGH Procedure: PCR 1.8 kb 2.0 kb

Validation of CGH Procedure: PCR + Nde I Digestion 2.0 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

False Positives False Positive RedsAverage Red/Green Ratio Red 5'-3': Identity Score (Upper Limit =44) YLR243W32981 YIL012W YKL215C5.559 YOR296W False Positive Greens Average Green/Red Ratio Green 5'-3': Identity Score (Upper Limit =49) YEL020W-A YBR162W-A YCR094W YGR153W YBR006WOff scale58 YLR174W YHR057C YLR087COff scale51 YBR034COff scale51 YNL015W YOR046COff scale50

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