ARTICLE |
Correspondence to:
Anton K. Raap, Lab. for Cytochemistry and Cytometry, Dept. of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. E-mail:
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Summary |
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Fluorescence in situ hybridization to DNA fibers (FiberFISH) is a high-resolution, wide-ranging physical DNA mapping method that finds increasing application in the study of pathological gene rearrangements. Here we present experiments designed to understand the nature of the discontinuous FISH signal patterns seen after FiberFISH. Use of a novel cisplatin-based chemical labeling method enabled us to produce intact biotin-labeled cosmid target DNA molecules. We monitored by immunofluorescence the fate of such cosmid targets during denaturation and hybridization. The same cosmid DNA labeled with digoxigenin by nick-translation was used to analyze the FISH probe signal distribution in a different color. The probe signals proved to be a subset of the target signals remaining after denaturation and hybridization. We argue that the discontinuity of probe signals in FiberFISH is mainly caused by loss of target DNA and limited accessibility due to in situ renaturation and attachment. Furthermore, we conclude that FISH sensitivity is determined by hybridization efficiency and not the ability to generate sufficient signal from small probes. (J Histochem Cytochem 48:743745, 2000)
Key Words: DNA mapping, FiberFISH, hybridization
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Introduction |
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D fluorescence in situ hybridization to DNA fibers (FiberFISH) (
Typically, large insert clones such as cosmids, bacterial, and yeast artificial chromosomes are used as hybridization probes in FiberFISH and specific hybridization to whole genomes or cloned DNA targets is recognized as linear or near-linear stretches of beads-on-a-string fluorescence signals. Factors such as loss and inaccessibility of target DNA due to either in situ renaturation or attachment to the glass substrate, as well as suppression of repeat sequences, have been put forward as contributing to the origin of the typical discontinuous FiberFISH pattern (
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Materials and Methods |
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Cosmid cAL24 (
FiberFISH results were imaged by digital fluorescence microscopy according to
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Results |
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Fig 1a shows images of biotinated target cosmid molecules before denaturation and hybridization and Fig 1b of target and probe cosmid signals after denaturation and hybridization. The overall fluorescence intensities of denatured and hybridized target cosmid molecules are much lower than those of undenatured targets. After denaturation alone, signal distribution and intensities were similar to those of target cosmids that were denatured and hybridized (not shown).
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Discussion |
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Because it is unlikely that inaccessibility for biotin detection reagents will decrease with denaturation, the reduction in the signal intensities of the target cosmids after denaturation can be accounted for by DNA loss.
When the distribution of the hybridization signals is compared with that of the target (Fig 1b), it is clear that there are stretches of target signals not bearing any hybridization signal and that there is no consistent signal distribution pattern. Because the present homologous cosmid DNA hybridizations were conducted without unlabeled Cot-1 DNA, it can be concluded that the typical beads-on-a-string FiberFISH pattern seen in Fig 1b does not originate from suppression of repeats but rather from technicalities in the FISH protocol. It should be noted, however, that in FiberFISH to whole genomes using large insert clones as probes in the presence of Cot-1 DNA for suppression of repeats, the sizes of tandemly repeated sequences may be such that they can contribute to the appearance of gaps. Consistent appearance of large gaps with FiberFISH to whole genomes has indeed been reported (
The fact that the hybridization signals are a subset of the remaining target signals strongly indicates that, next to DNA loss, accessibility of target DNA is a main determinant of hybridization efficiency. This inaccessibility may be due to the process of in situ renaturation or to physical attachment to the glass surface per se.
We conclude that the relative scarcity of the hybridization signals, as seen in these experiments, is a consequence of DNA loss during denaturation, in situ renaturation of target, and limited reagent accessibility due to attachment. This implies that a target fragment has a random chance of obtaining a FISH signal. This randomness explains the absence of consistent beads-on-string signal patterns in the present experiments. As noted previously, suppression of repeat sequences may contribute to the appearance of gaps in FiberFISH to genomic DNA.
Previously, we have detected sub-kb targets by genomic FiberFISH and have noted a decrease in efficiency of detection as target sizes became smaller. Nevertheless, the FISH intensities of the smallest target tested (202 bp) were detected with an efficiency of about 30%, indicating that smaller sequences should be detectable as such, even with the standard indirect FISH techniques used (
It is noteworthy that FISH with cosmid probes to metaphase and interphase cells leads to single signals at the target sites with intensities comparable to those of a single signal in a cosmid FiberFISH track, indicating that, because of a higher level of inaccessibility, hybridization efficiencies in interphase cells and chromosomes are much lower than those of FiberFISH, perhaps by as much as a factor of 10.
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Acknowledgments |
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We thank Kreatech Diagnostics for the biotinULS reagent.
Received for publication November 7, 1999; accepted February 23, 2000.
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Literature Cited |
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