High-resolution `Fiber-FISH`: The generation of barcodes along large genes and the study of replication structures in human DNA
- Leiden Univ. (Netherlands); and others
Fluorescent In Situ Hybridization (FISH) is a powerful technique enabling the simultaneous study of multiple targets. FISH covers a wide range of genomic resolutions and recently developed spreading techniques bring the resolution of FISH mapping to the level of the DNA double helix itself. We have successfully used these `Fiber-FISH` approaches, in combination with digital image analysis, as tools to assess cosmid contigs, YAC maps, YAC chimerism, the sizing of gaps/overlaps and study of replication structures in human DNA. The size estimates obtained are highly accurate and reproducible down to 0.3 {mu}m or 1 kb, the theoretical light microscopic resolution. Fiber-FISH on human cells allowed mapping and ordering of sequences at distances ranging from 1 to 400 kb. For two large genes, thyroglobin (330 kb) and dystrophin (2.3 Mb), linear, multi-color bar codes were derived using adjacent and overlapping cosmids. This approach should ultimately enable the rapid identification of gene rearrangements. Furthermore, we developed a Fiber-FISH protocol using cosmid probes on YACs in decondensed yeast DNA. The high resolution, combined with the high efficiency and strong signals obtained, enabled both the establishment of detailed linear maps and the direct visualization of replication `forks` and `bubbles`. The multi-color patterns directionally orient the replication process and identify sequences in cloned human DNA which function as replication origins in yeast. Replication intermediates were also observed in unsynchronized human cells, suggesting that Fiber-FISH allows mapping of replication origins along megabase regions of DNA. This opens exciting possibilities to study mammalian replication, its timing, its delineation in replication zones and its potential relation with processes as X-inactivation and genomic imprinting.
- OSTI ID:
- 134398
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-1132
- Journal Information:
- American Journal of Human Genetics, Vol. 55, Issue Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BASIC STUDIES
44 INSTRUMENTATION
INCLUDING NUCLEAR AND PARTICLE DETECTORS
GENES
SIZE
DNA HYBRIDIZATION
GENETIC MAPPING
GENE MUTATIONS
FLUORESCENCE
ACCURACY
RELIABILITY
RESOLUTION
EFFICIENCY
CONTIGS
DETECTION
DNA
DNA REPLICATION
STRUCTURE-ACTIVITY RELATIONSHIPS
YEASTS
PROBES
DNA-CLONING