Utilization of FISH in positional cloning: an example on 13q22.

Abstract

in positional cloning the initial assignment of a gene to a specific chromosomal locus is followed by physical mapping of the critical region. The construction of a high-resolution physical map still involves considerable effort. However, new high-resolution fluorescence in situ hybridization (FISH) techniques have facilitated this process substantially. Here we summarize a strategy that combines a spectrum of FISH techniques [metaphase, interphase, mechanically stretched chromosomes (MSCs), and fiber-FISH on free chromatin] for the construction and characterization of a high-resolution physical map for a positional cloning project. The chromosomal region 13q22, containing the locus of the variant form of the neuronal ceroid lipofuscinosis (vLINCL, CLNS) disease, serves here as an example for this process. We used metaphase FISH to exclude positionally a candidate gene, to refine the locus to 13q22, and to analyze the possible chimerism of the YACs in the region. Both metaphase and interphase FISH techniques were applied to determine the low-resolution distances between the restricting markers. FISH using MSCs confirmed the centromeric-telomeric order of the clones and facilitated the estimation of the size of the gaps between the clones. Finally, fiber-FISH was found to be the method of choice for the construction of an accurate high-resolution map of the contig established over the restricted region. Thus, FISH techniques in combination with genetic mapping data enabled the refinement of the initial 4-cM region to a high-resolution map of only 400 kb in length. Here the FISH strategy replaced the need for many laborious traditional physical mapping methods, e.g., pulsed-field gel electrophoresis.

Localization of FCGR1 encoding Fcγ receptor class I in primates: molecular evidence for two pericentric inversions during the evolution of human chromosome 1

“…While others have mapped some of the FCGR1 genes to either 1q21.1 (de Wit et al, 1993) or 1q21.2 → q21.3 (Takai et al, 1994), we did not attempt to sublocalize FCGR1 in this study. However, the fact that seven human chromosomes 1 yielded separate signals for FCGR1A and FCGR1C at 1q21 leads us to estimate that these genes are located approximately 500-1,000 kb apart (Lichter et al, 1990;Laan et al, 1996).…”

Analysis of Tie receptor tyrosine kinase in haemopoietic progenitor and leukaemia cells

“…The cloned BamHI-XhoI fragments of Ang-1 and Ang-2 were used for screening of human genomic bacterial artifical chromosome (BAC) clones (Genome Systems Inc., St Louis, Mo., U.S.A.). The clones were labelled with either biotin-11-dUTP (Sigma Chemicals) or digoxigenin-11-dUTP (Boehringer Mannheim) by nick translation according to standard protocols and hybridized with the metaphase spreads as previously described (Pinkel et al, 1986;Lichter et al, 1988;Laan et al, 1996). The co-hybridization of the angiopoietin probes with the c-myc (Alitalo et al, 1983) and hCAT-2 (Lauteala et al, 1997) plasmid probes, as well as the two-colour FISH were performed and analysed as described by Heiskanen et al (1996).…”

Application of fiber-FISH in physical mapping of Arabidopsis thaliana

“…This technique can be used to analyze overlapping clones (Heiskanen et al 1994), to detect chromosomal rearrangements (Heiskanen et al 1995b), to determine the physical distance between genes and their 5′ to 3′ orientation (Heiskanen et al 1995a), to measure sizes of long DNA loci (Shiels et al 1997), and eventually, to expedite positional cloning (Laan et al 1996). Fransz et al (1996) successfully adopted the fiber-FISH technique for plant species.…”