FISH metaphase sequences

In summary, when cultivating these cell lines in the absence of MTX, unique and characteristic integrations were found in 95-99% of metaphase spreads. We continued to cultivate these cell lines for extended periods in the absence of MTX, and occasionally performed FISH analyses. We found that the chromosomal structures described above were stable within the observation period (a minimum of 60 days, and in one case of 160 days). Cytogenetically, these observations indicate a high degree of genetic stability of chromosomally amplified sequences in the absence of MTX. Equivalent observations as those presented above have been made recently by Kim and Lee [106]. 

Most satellite DNA is composed of repeats of 14-500 base pairs in tandem repeats of 20-100 kb. In situ hybridization studies with metaphase chromosomes have localized these satellite DNAs to specific chromosomal regions. In most mammals, much of this satellite DNA lies near centromeres, the discrete chromosomal regions that attach to spindle microtubules during mitosis and meiosis. Satellite DNA is also located at telomeres, the ends of chromosomes, and at specific locations within chromosome arms in some organisms. These latter sequences can be useful for identifying particular chromosomes by fluorescence in situ hybridization (FISH), as Illustrated in Figure 10-5. 

DNA-QD conjugates have also been used as luminescent labels in fluorescence in situ hybridization (FISH) assays. The FISH assay is based on the denaturation of genomic DNA and the consecutive hybridization with a fluorescent-labeled DNA sequence, in order to visualize the presence or absence of specific DNA sequences in the chromosomes. For example, a QDs-based FISH labeling method was used to detect the Y chromosome in human sperm cells [74], and also the human metaphase chromosome [150]. The FISH technique was also used for the multiplex cellular detection of different mRNA targets [151], and to visualize a pUClS plasmid inside the Escherichia coli HBlOl bacterium [152]. 

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