The hybridization site of a DNA probe was detected using a scanning electron microscope (SEM), modifying the standard in situ hybridization (ISH) method.The experiments were performed on human metaphases obtained from lymphocyte cultures of human peripheral blood. The libraries and probes used were: 1-chromosome library for the painting of chromosome 1 (wcp 1), an alphoid centromere-specific probe of chromosome 8 (pZ8.4), and the yeast artificial chromosome (YAC) 964-C10 mapped at band p13 on chromosome 12. These probes were labeled by nick translation with biotin and displayed with a gold-conjugated anti biotin goat antibody. The gold signal was amplified by silver enhancement. The chromatides appeared as packages of thin filaments 120 nm high; some of them collapsed, probably due to ISH procedures. All the probes were clearly detected as small gold particles grouped on the surface of the target chromosomes and chromosome sites. Thus, this procedure is useful to clarify the positional relationship between the chromatin filaments and the probe.

In situ hybridization by scanning electron microscopy for painting, centromeric, and YAC localization

REGUZZONI, MARCELLA;PROTASONI, MARINA;MASERATI, EMANUELA;PRESSATO, BARBARA;RASPANTI, MARIO
2005

Abstract

The hybridization site of a DNA probe was detected using a scanning electron microscope (SEM), modifying the standard in situ hybridization (ISH) method.The experiments were performed on human metaphases obtained from lymphocyte cultures of human peripheral blood. The libraries and probes used were: 1-chromosome library for the painting of chromosome 1 (wcp 1), an alphoid centromere-specific probe of chromosome 8 (pZ8.4), and the yeast artificial chromosome (YAC) 964-C10 mapped at band p13 on chromosome 12. These probes were labeled by nick translation with biotin and displayed with a gold-conjugated anti biotin goat antibody. The gold signal was amplified by silver enhancement. The chromatides appeared as packages of thin filaments 120 nm high; some of them collapsed, probably due to ISH procedures. All the probes were clearly detected as small gold particles grouped on the surface of the target chromosomes and chromosome sites. Thus, this procedure is useful to clarify the positional relationship between the chromatin filaments and the probe.
scanning electron microscope (SEM); situ hybridization (ISH) method; chromosome
Reguzzoni, Marcella; Protasoni, Marina; Maserati, Emanuela; Pressato, Barbara; Manelli, A; Raspanti, Mario
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11383/1496367
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