Fiber FISH
FISH conducted on Extended Chromatin Fiber Preparations
In mapping DNA fragments of interest by conducting Fluorescence In Situ Hybridization (FISH) on chromosomes, signals within a distance of several Mbps (Mbps=1,000,000 base pairs) are indistinguishable from each other because of the multifold structure of DNA strands in the metaphase chromosomes. The resolution of signals improves if the chromosomes are used before they progress to full condensation. What can we do when we want to map more adjacent DNA clones? The characterization of entire genome DNA sequences will resolve the problem of creating a map in scale of one base pair, but is extremely time-consuming.
The human genome is calculated to contain 30,000-100,000 genes, that is, 1,200-4,000 genes per chromosome on average. The genes of 10-15 kbs (kbs=1,000 bases) of average size line up on DNA strands at intervals of 40-45 kbs. New procedures are required in order to produce a detailed map of DNA fragments containing these genes.
I discuss here two ways to overcome the resolution limits found when using FISH on metaphase chromosomes. Mapping under 1 Mbps resolution is available by using stretched chromatin (DNA) fibers.
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Fig. 1 Resolution of FISH |
Mapping Under 1 Mbps Resolution
- FISH on interphase nuclei
A high-resolution map can be created by using the interphase nuclei as chromatin fibers extending into the nuclei because they stretch much more compare to that in the metaphase chromosomes (Fig. 1). This procedure is can be used to map DNA clones at a distance of 50-500 kbs, however its accuracy is somewhat inaccurate because the degree of extension of chromatin fibers varies from place to place. - FISH on extended chromatin fiber preparation
Higher resolution for detection of signals is obtained by artificially extending chromatin fibers on a slide glass before DNA hybridization (Fig. 1).
Procedure of Fiber FISH
- Cells are spread and air-dried on a slide glass
- Cells on a slide glass are lysed by detergent (Triton X-100) in a jar
- Chromatin fibers attach to the slide glass when it is slowly removed from the jar
- The preparation is fixed with ethanol
- High resolution FISH at a distance of 5-700 kbs becomes possible
- The probes are hybridized following standard procedure
Fiber FISH is currently used to order a series of DNA fragments cloned from chromosomes and to estimate the distance between these clones. However, DNA strands must still be examined at the submicroscopic level for more detail. The difficulty of submicroscopic measurement makes tracking and distance measurements unclear. Current research is exploring potential methods of simultaneous detection of signals and DNA strands. Scanning probe microscopy is a promising candidate.
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