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Asrm2008/Oligo-FISH allows enumeration of 18 chromosomes in 4 successive hybridizations performed in 1 day
- 1. Oligo-FISH allows enumeration of 18 chromosomes in 4 successive hybridizations performed in 1 day Joan Aurich-Costa, Leah Zamechek, Carrie Ng, Susan Selvaggio and Sean Bradley Cellay, Inc., Cambridge, MA 02139
- 2. Oligo-FISH allows enumeration of 18 chromosomes in 4 successive hybridizations performed in 1 day Joan Aurich-Costa, Leah Zamechek, Carrie Ng, Susan Selvaggio and Sean Bradley Cellay, Inc., Cambridge, MA 02139 20
- 4. S/N for 6 successive oligo-FISH
- 6. 4 Successive hybridizations on same cells FISH 1: X-R, Y-G, 15-Y, 17-A, 20-P FISH 2: Yq12-P, 1q12-Y, 4-G, 10-R, 16q12-A FISH 3: 3-R, 7-A, 8-Y, 11-G, 16-P FISH 4: 2-R, 6-A, 9q12-Y, 12-P, 18-G
- 10. FISH 1: 3-R, 7-A, 12-Gd, 16-G 12 12 3 3 7 16 7 16
- 11. FISH 2: 2-R, 13-21-Gd, 18-A, 20-G 12 12 3 3 7 16 7 16 18 18 20 20 21 21 13 13 2 2
- 12. FISH 3: 6-A, 8-G, 9q12-Gd, 11-R 12 12 3 3 7 16 7 16 18 18 20 20 21 21 13 13 2 2 9 11 11 8 8 6 6
- 13. FISH 4: X-R, Yq12-G, 15-Gd, 17-A 12 12 3 3 7 16 7 16 18 18 20 20 21 21 13 13 2 2 9 11 11 8 8 6 6 17 17 Y X 15 15
- 14. FISH 5: 1q12-A, 4-G, 10-R, 14-22-Gd 12 12 3 3 7 16 7 16 18 18 20 20 21 21 13 13 2 2 9 11 11 8 8 6 6 17 17 Y X 15 15 4 14 22 4 14 10 10 22 1 1
- 15. FISH 6: Spectrum Orange labeled RB1 gene on 13 20 chromosomes are identified + 14-22 12 12 3 3 7 16 7 16 18 18 20 20 21 21 13 13 2 2 9 11 11 8 8 6 6 17 17 Y X 15 15 4 14 22 4 14 10 10 22 1 1
- 16. 6 successive hybridizations, 4 visible colors, 21 probes Signal/Noise
- 17. 6 successive hybridizations, 4 visible colors, 21 probes Sensitivity
- 20. Future considerations Empire Genomics will provide the BAC probe kit for enumerating chromosomes 5, 19, 21 and 22 24 Chromosomes / 24 hours
- 21. The FISH Team Joan Aurich, Ph.D. Susie Selvaggio Carrie Ng Leah Zamechek Sean Bradley, Ph.D.
Editor's Notes
- First, I would like to thank you for letting us present our FISH probe developments in this meeting. Last year we presented in this meeting the development of the Oligo-FISH probes and specially one of their most amazing characteristics which is their 5 minutes hybridization. Because of the low stringency required and the short hybridization time we wanted to explore how they behave in successive FISH
- Actually, when we submitted the abstract we only had 18 chromosomes, but I will show you results with 20 chromosomes…
- When traditional probes, derived from BACs are used, no more than 3 hybridizations can be performed on the same slide without compromising the integrity of the DNA. Furthermore, each hybridization takes between 4 to 6 hours or they are performed overnight, limiting a lot the number of hybridizations that can be performed if a result is expected in 24 hours.
- Because the oligo FISH probes require less stringency and a very short hybridization time, we expected that more than 3 hybridizations could be performed without damaging the DNA. For this purpose the same slide was hybridized 6 times in a row with the same probe cocktail. We calculated the signal to noise ratio to evaluate the lost of signal due to the damage caused to the DNA. There was just a little lose of signal at each hybridization. Despite the 6 hybridizations the signal to noise ratios were always maintained at more that 2, showing that at least 6 hybridizations could be performed on the same slide.
- At this time we had 20 probes covering 18 chromosomes. In order to try successive hybridizations with all the probes we had developed we started composing 4 kits of 5 probes each. This should allow us to enumerate 20 loci covering 18 chromosomes. Obviously we had to use our Far Red fluor as the 5 th color…
- And this is the result. After 4 hybridizations the DNA remain intact and all the signals were totally observable.
- Although we performed this experiment on one day, we calculated all the times required in detail including taking the pictures of 3 cells that should correspond to the time of analysis. Since we were using peripheral blood slides, because we needed to verify the specificity on chromosomes, first we had to pretreate the slides. It took about 1hour and 20min, but these treatments are only performed once, and does not apply to all cells. After every hybridization the slides need to be cleaned, to remove the oil and the antifade, and need to be dehydrated again. Although this step is not required for the first hybridization it will be required in the successive ones. The initial denaturation was of 2 min, and only 1 min in the successive hybridizations, but with the dehydration steps this always takes about 14-15 min. 5 minutes hybridization, and the washes about 15 min Finding the cells on the slide using the micrometric stage positioning (not automatic), and taking the pictures on each channel took about 10 min. All together, cleaning, denaturation, hybridization and analysis took about 1 hour. So 20 loci in 4 successive FISH where enumerated in only 4 hours. This result encouraged us to try to perform more hybridizations in an 8 hours working day… Like…We could perform 5 hybridizations with only 4 visible colors, for those among you that want to see the signals with their human eyes =)
- Usign the sequence the chromosomes that we cannot develop specific probes are 5 and 19, 13 and 21 and, 14 and 22. Finally we had developed probes for chromosomes 13 and 21, and 14 and 22. Although, we know that for diagnostic we should use probes specific for one single chromosome, If after we hybridize with probes specific for one of the chromosomes of each pair we can deduce the number of the other chromosome. For example if we have 4 signals for the probe 13-21, and then we hybridize a BAC for chromosome 13 that results in 2 signals it means there is 2 chromosomes 13 and 2 chromosomes 21.
- These are the panels we hybridized sequentially on the same slide. Since we observed a slight decrease in signal to noise ratio we put the most intense probes in the later hybridizations and the weakest probes in the first hybridizations. In order to verify that the DNA is still accessible we performed a 6 th hybridization with a BAC probe containing RB1 gene on chromosome 13 that will also help us enumerate the number of chromosomes 21.
- And here are the results of first hybridization, in yellow…
- Second hybridization with yellow arrows, and we keep the arrows from the first hybridization in white…
- Third hybridization…
- 4 th hybridization
- 5 th hybridization
- And finally the last hybridization with a BAC probe specific of chromosome 13. As you can see all the chromosomes and the nuclei still maintain their morphology and show nice signals… If we were unable to see the chromosomes as for the interphase nuclei, knowing now that there are two signals for chromosome 13, and having seen 4 signals with the 13-21 probe we can conclude that there are two 13s and 2 21s…
- As you can see in this graph all the probes showed a signal to noise ratio superior to 2. 2 is the threshold to be able to comfortably differentiate a hybridization signal from the background.
- We also calculated for each probe the sensitivity, all the probes but 3 reached sensitivities superior to 98%. The scale of this graph is only between 95% and 100% to show clearly the differences... The lowest sensitivity was reached by the BAC probe although it was 97% that is still quite high,
- Regarding the time necessary to perform FISH and analyze 3 cells at every step it was confirmed to about 1 hour. The 5 oligo FISH were performed in 5 hours, obviously for the BAC probe we hybridized overnight, but it still means that in a frame of 24 hours all these chromosomes could be analyzed. However, in the future we will shorten this time since it could probably be hybridized in 4 to 6 hours.
- Please come to visit us at Booth 119 Thank-you very much for your attention