Notes

1. Karyotyping medium is based on RPMI-1640 basal medium, supplemented with L-glutamine, fetal bovine serum, and antibiotics (penicillin and streptomycin) and containing phytohemagglutinin.

2. Prewarm the Colcemid and the hypotonic solution in the incubator at 37°C.

3. Caution: Colcemid can be dangerous, so handle with care. Colcemid is a mitotic spindle inhibitor. If splashed on skin, rinse immediately and seek medical help.

4. Caution: The formamide is toxic and may cause harm to the unborn children. Harmful by inhalation, by contact with skin, or if swallowed. Therefore, the formamide solution needs to be prepared and used by taking precautionary measures for safety work.

5. The primers used to amplify in si?«-specific exons of DMD gene need to be of 20 to 22 bp and have the annealing temperature ranging from 55 to 58°C.

6. All reagents are stable at -20°C. Repeated freezing and thawing should be avoided. The PRINS-modified mixture must be freshly prepared before each use.

7. To prepare 0.5 M EDTA, pH 8.0, dissolve 186.1 g of EDTA disodium salt in 800 mL of distilled water and mix on magnetic stirrer using slight heating. Adjust the pH at room temperature and then add distilled water to final volume of 1000 mL.

8. Triton X-100 is a dense liquid; therefore, a tip with large lumen need to be used. Mix and dissolve completely by using magnetic stirrer.

9. For FITC-conjugated antidigoxigenin antibody, the reconstituted solution is stable at -20°C. To avoid repeated freezing and thawing, the solution must be stored in aliquots at -20°C protected from light.

10. The hypotonic solution should not be in contact with the cells for more than a total of 27 min. Excess exposure may cause rupture of the white blood cells.

11. The fixative solution must be made fresh.

12. Thirty minutes is a minimum. It is possible to keep samples in the refrigerator overnight.

13. If the annealing and elongation reactions are performed using hot plate, it is necessary to increase the temperature setting of 2°C to avoid the dispersion of temperature on glass.

14. For the acquisition of images by confocal laser microscopy, FITC and PI need to be excited with the blue (488 nm) and green (514 nm) line of the argon ion laser, respectively. Thereafter, serial optical section of FITC signal, performed on z-axis and merged with the corresponding PI images, will be elaborated and reconstructed as a three-dimensional projection.

Acknowledgments

We thank Dr. Carmela Trimarchi for technical assistance and her for the helpful and constructive comments. This work was supported by CNR grant.

References

1. Sellner, L. N. and Taylor, G. R. (2004) MLPA and MAPH: new techniques for detection of gene deletions. Hum. Mutat. 23, 413-419.

2. Muntoni, F., Torelli, S., and Ferlini, A. (2003) Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol. 2, 731-740.

3. Mandel, J. L. (1989). Dystrophin. The gene and its product. Nature339, 584-586

4. Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., Nguyen, P. N., and Caskey, C. T. (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16, 11,141-11,156.

5. Den Dunnen, J. T., Grootscholten, P. M., Bakker, E., Blonden, L., Ginjaar, H. B., Wapenaar, M. C., et al. (1989). Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am. J. Hum. Genet. 45, 835-847.

6. Beggs, A. H., Koenig, M., Boyce, F. M., and Kunkel, L. M. (1990). Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum. Genet. 86, 45-48.

7. Koenig M., Monaco A. P., and Kunkel L. M. (1988). The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53, 219-226.

8. Muntoni, F. and Strong, P. N. (1989) Transcription of the dystrophin gene in Duchenne muscular dystrophy muscle. FEBS Lett. 252, 95-98.

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