[Extending the capabilities of human chromosome analysis: from high-resolution banding to chromatin fiber-FISH].
The rapid development in human chromosome studies during the past 2 decades has opened up a variety of new avenues in both basic and clinical human genetics. This has been due to the extended resolution of chromosome analysis especially by the introduction of high-resolution banding and fluorescence in situ hybridization (FISH) techniques. High-resolution banding methods using elongated chromosomes from cells at early mitotic stage can be divided into two main categories: one involves S-phase synchronization using methotrexate or thymidine with subsequent release from the block, and the other involves the application of DNA-binding agents, such as ethidium bromide, which inhibit mitotic chromosome condensation. High-resolution band analysis facilitates refined determination of the breakpoints on rearranged chromosomes, and thus minute deletions, small translocations and other subtle chromosome mutations can readily be detected and identified. It has also proved to be valuable for accurate gene mapping and comparative cytogenetics. Each of the high-resolution bands consists of DNA molecules as long as approximately 3Mb on the average, and this resolution limitation has been overcome by the introduction of the FISH method, which enables chromosome and genomic analyses to be carried out at the DNA molecule level. For FISH analysis, appropriate DNA probes of various insert sizes can be chosen for different purposes, and FISH with specific probes permits high-resolution analysis of chromosome DNA constitution at particular loci. For instance, it is possible to detect and map unique sequences more than 1 kb in size on mitotic chromosomes, and to identify cryptic chromosome rearrangements not recognizable even by high-resolution band analysis. Better resolution of breakpoint determination can also be expected by FISH using an adequate series of DNA clones arrayed on the physical map within the relevant chromosome region. An even better genomic resolution by FISH can be obtained by utilizing free chromatin DNA fibers released from interphase nuclei as a hybrization target. The FISH signals on the chromatin fiber preparations are shown to be "linear", unlike the standard FISH signals that have a "dot" appearance, and the resolution attainable should be comparable to analysis of a straight DNA double helix itself. The degree of resolution in the so-called fiber-FISH method ranges from a few to 300 kb, and thus the method can readily be applied to the high-resolution assessment of ordering and overlapping of isolated DNA clones from a specific chromosome region, as well as sizing of gaps between the clones. Fiber-FISH may prove most valuable for the precise determination of breakpoints at the kilobase level, and also for identifying minute deletions and duplications. By presenting some examples of our own recent works, this review attempts to summarize the current contribution of the above strategies in mediating the two separate ranges of resolution achieved by the classical chromosome and molecular DNA analyses.
Ikeuchi
T
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Department of Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, Japan.
D002843:Chromatin; D002871:Chromosome Banding / Q000379:methods*; D019457:Chromosome Breakage; D002874:Chromosome Mapping; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D014178:Translocation, Genetic
D002843:Chromatin