Related Experiment Videos
[A FISH technique for simultaneous detection of fluorescent R-band and in situ hybridization signals]
1Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005 P. R. China.
Objective:
To develop a simple method for detecting DNA probes directly on R-banded chromosomes.
Methods:
After sixty-seven hours culture, human peripheral blood lymphocytes were synchronized for 5 to 6 hours by adding Hoechst 33258 and BUdR, and then arrested by standard cytogenetic procedures. The slides were mounted with 2 x SSC and exposed with a 20W UV light which was about 10cm above the slides for 20 min at 75 degrees C. The biotinylated probes, such as the cosmid and YAC clones on 5p specific region and pBamX7, were hybridized on to the slides. After washing, the slides were treated with avidin-FITC and amplified with additional layer of biotinylated anti-avidin and avidin-FITC, and counterstained with propidium iodide in an antifade solution. Fluorescent signals and R-bands were observed simultaneously under Olympus BX 60 fluorescence microscope equipped with a WIB filter.
Results:
The chromosomal location of the greenish-yellow signals could be directly identified on the R-banded chromosome background.
Conclusion:
This method can serve as a rapid and precise system for chromosomal localization of DNA markers.