High-resolution FISH of stretched chromosome fibers

Abstract

High resolution fluorescence in situ hybridization (FISH), so-called FIBER-FISH, was first described by Heng et al. (1992) for long stretched DNA-fibers and was later subjected to modifications by other investigators (Fidlerova et al. 1994, Heiskanen et al. 1996). The resolution using cosmids on DNA fibers was restricted by the length of the visible, decondensed DNA strand up to 150 to 450 kb. Using earlier protocols, it was not possible to obtain a coherent DNA strand showing a longer section of cosmids in direct orientation. However, freshly prepared lymphocyte suspension could be used (for lymphocyte preparation see Arnoldus et al., 1990, Verma et al., 1989).This method described here is simple, but very efficient. It allows the presentation of cosmids over a region up to 8 Mb in a linear orientation. Thus, the visualization of the transcriptional orientation of genes is possible within a bigger range. Moreover, the modified treatment allows the use of archival suspensions of cultured lymphocytes and is also available for YACs. The main steps are a special treatment of up to two year old lymphocyte suspensions, the disruption of nuclei by a shortened PBS incubation previous to alkaline treatment and the release of chromosome fibers from interphase nuclei by special handling of the slides. With these modifications, we were able to obtain released chromosome fibers, which were suitable for a high resolution two-color FISH. This method has been specially tested for lymphocyte nuclei (Fuchs et al., 1997).