FISH Probes Design and Protocols

Introduction to FISH Probes

FISH Probes Applications

FISH Probes Design/Protocol

FISH Probes Literature

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FISH Probes Design / Protocol

Design Guidelines for FISH Probes
1. Design multiple 24 to 30mer probes. Avoid stretches of more than 3 G or C bases.
2. To impart exonuclease resistance substitute 3-4 bases at the 5' and 3' end with 2'F bases. The 2' F bases imparts resistance to exonuclease degradation and increases duplex stability by 4-6 degrees.
3. Several internal bases can be substituted with 5me dC and 2 Amino dA to further increase duplex stability.
4. Affinity ligands such as Digoxigenin or Biotin or fluorescent dye e.g Cy3, Cy5 or any other can be labeled at the 3' and 5' end. Multiple internal sites can also be labeled with affinity ligands or fluorescent dyes to increase sensitivity.
5. Multiple dye sites should be spaced apart by 10 or more bases.
6. The above guidelines are for all initial FISH probe design. Design rules may have to be established empirically for very specific or novel assay settings, but following the above recommendations will provide a good start.

References

1. Milligan, J.F., Matteucci, M.D. and Martin, J.C. (1993) Current concepts in antisense drug design. J. Medicinal Chem. 36:1923-1937.
2. Helene, C., Toulme, J. (1990) Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim. Biophys. Acta. 1049: 99-125.
3. Weintraub, H. M. (1990) Antisense RNA and DNA. Sci. Amer. 262:40-46.
4. Iyer, R.P., Egan. W., Regan, J.B and Beaucage, S.L. (1990) J. Am. Chem. Soc.112; 1253-1254.
5. Wagner, R.W., Matteucci, M.D., Lewis, J.G., Gutierrez, A.J., Moulds, C. and Froehler, B.C. (1993) Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines. Science 260:1510-1513.
6. Hertoghs, K.M.L., Ellis, J.H. and Catchpole, I.R (2003) Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA. Nucl. Acids Res.31 (20): 5817-5830.
7. Cotton, M., Oberhauser, B., Burnar, H. et al. (1991) 2'O methyl and 2'O ethyl oligoribonucleotides as inhibitors of the in vitro U7 snRNPdependent messenger-RNA processing event. NAR 19:2629.
8. Singh, S.K., Nielsen, P., Koshkin, A.A. and J. Wengel, Chem. Comm., 1998, (4), 455-456.
9. A.A. Koshkin, A.A., Singh, S.K., Nielsen, P., Rajwanshi, V.K., Kumar, R., Meldgaard, M., Olsen, C.E and Wengel, J. (1998) Tetrahedron 54:3607-3630.
10. Kv�rn�, L. and Wengel, J. (1999) Chem. Comm., 7:657-658.
11. Petersen, M and Wengel, J. (2003) Trends in Biotechnology 21(2): 74-81.
12. P.A. Giannaris, P.A. and Damha, M.J (1993) Nucleic Acids Research, 21:4742-4749.
13. Bhan, P., Bhan, A., Hong, M.K., Hartwell, J.G., Saunders, J.M and Hoke, G.D (1997) Nucleic Acids Res, 25:3310-3317.
14. Coleman, R.S. and Kesicki, E.S (1994) J. Amer. Chem. Soc., 116:11636-11642. 15. John SantaLucia, Jr. (1998) Proc. Natl. Acad. Sci. 95 1460-1465.