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

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 Red = wt Green = Evolved No binding

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

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

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

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

21. YeaStar Genomic DNA Isolation 0.5 mL yeast cells1.5 mL yeast cells Concentration (ng/ µ L) 31.743.1 Absorption at 260 nm 0.6340.861 Ratio 260/280 1.751.86

22. YeaStar + ZR Genomic DNA kit wt S288C Evolved L  42K Concentration (ng/ µ L) 12235.2 Absorption at 260 nm 0.2450.703 Ratio 260/280 1.911.90 >12 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 kitPromega kit Percent yield 90.6% 74.9%

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

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

27. Genisphere CGH Procedure: Minimize Background Before AfterBeforeAfter

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 ChromosomeBiological Process Molecular Function Cellular Component YAR020C0.4820.3711 unknown YBR123C0.5030.4202 Transcription initiation Pol III promoter RNA poly. III transcription factor Transcription factor TFIIIC complex YBR169C0.3550.3582 Protein foldingHeat shock protein activityunknown YDL042C0.5020.4204 Chromatin silencing at telomere Histone deacetylase activity nucleolus YDR087C0.4800.3574 rRNA processingunknown YDR131C0.5480.3714 Ubiquitin-dependent protein catabolism Protein binding activityUbiquitin ligase complex YDR211W0.4900.2044 Translation initiationTranslation initiation factor activity Ribosome YER102W0.3830.2455 Protein biosynthesisStructural constituent of ribosome Cytosolic small ribosomal subunit (sensu Eukarya) YER104W0.4390.3905 Neg. reg. of DNA transposition unknown YGR072W0.4410.2177 mRNA catabolismunknownCytoplasm YOL162W0.5460.44015 TransportTransporter activityMembrane YPR049C0.5000.40416 Protein-vacuolar targetingunknownExtrinsic to membrane

31. Validation of CGH Procedure: PCR 1.8 kb 2.0 kb

32. Validation of CGH Procedure: PCR + Nde I Digestion 2.0 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 RedsAverage Red/Green Ratio Red 5'-3': Identity Score (Upper Limit =44) YLR243W32981 YIL012W12.863 YKL215C5.559 YOR296W23.649 False Positive Greens Average Green/Red Ratio Green 5'-3': Identity Score (Upper Limit =49) YEL020W-A5555.578 YBR162W-A5524.966 YCR094W393.560 YGR153W2652.560 YBR006WOff scale58 YLR174W138.853 YHR057C191.753 YLR087COff scale51 YBR034COff scale51 YNL015W277.550 YOR046COff scale50

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