Oligo-FISH): New Strategy For Enumerating Chromosomes In Interphase Nuclei Joan Aurich-Costa, Peter Keenan, Leah Zamechek and Sean Bradley Cellay, Inc., Cambridge, MA 02139

1. Oligo Fluorescence In Situ Hybridization (Oligo-FISH): New Strategy For Enumerating Chromosomes In Interphase Nuclei Joan Aurich-Costa, Peter Keenan, Leah Zamechek and Sean Bradley One Cell Systems, Inc., Cambridge, MA 02139 Support: NIH grant: 1R43 HD052597-01 NSF grant: IIP-0724876

4. Single Oligo-FISH Specificity Assessment

6. Signal/Noise ratio determination Segmentation DY549 DAPI+signal

7. All Probe Cocktails Labeled With DY549 0.06 3.61 Chr. 17 0.04 2.40 Chr. 20 0.03 2.17 Chr. 15 0.04 2.83 Chr. Y 0.05 3.40 Chr. X SEM S/N Probe

8. Conventional 5 Color FISH Blue Green Gold 350 400 450 500 550 600 650 700 750 Red Aqua

9. New Fluors DY405 DY490 DY549 350 400 450 500 550 600 650 DY590 DY415

10. Chr Y Probe Labeled With All Fluors Signal to Noise Ratio Assessment 0.11 3.80 DY590 0.07 2.30 DY649 0.09 4.20 DY560 0.07 4.30 DY549 0.06 2.10 DY505 0.03 2.50 DY490 0.90 3.90 DY415 0.05 1.30 DY405 SEM S/N Dye

11. DY490 DY549 400 450 500 550 600 650 700 750 DY649 DY415 5 Fluors – 4 Visible, 1 Far Red DY590

12. Which Color Per Probe? 2.17 Chr. 15 2.40 Chr. 20 2.83 Chr. Y 3.40 Chr. X 3.61 Chr. 17 S/N Probe 2.50 DY490 2.30 DY649 3.80 DY590 3.90 DY415 4.30 DY549 S/N Dye

13. Final Probe Combination Optimum Hybridization Time 5 Minutes

14. FISH Time Comparison ~15min ~4h 10min ~16h 25min Total time 5min 5min 20min Washes 5min 4hours Overnight (16h) Hybridization 2-5min 2-5min 2-5min Denaturation Oligo-FISH Fast-FISH Traditional FISH

15. Blastomere FISH

16. Sperm FISH

17. [email_address] www.cellayinc.com