Chapter 17
High Resolution FISH of Stretched Chromosome Fibers
BERND RAUTENSTRAUSS and CHRISTINA FUCHS
Introduction
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 aI., 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 aI.,
1997).
~ Bernd Rautenstrauss, Institut rur Humangenetik, Schwabachanlage 10, Erlangen,
91054, Germany (phone +49-9131-8532352; fax +49-9131-209297;
e-mail berndwr@humgenet.uni-erlangen.de)
Christina Fuchs, Institut rur Humangenetik, Schwabachanlage 10, Erlangen, 91054,
Germany
PROTOCOL
High Resolution FISH of Stretched Chromosome Fibers
BERND RAUTENSTRAUSS and CHRISTINA FUCHS
Introduction
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 aI., 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 aI.,
1997).
~ Bernd Rautenstrauss, Institut rur Humangenetik, Schwabachanlage 10, Erlangen,
91054, Germany (phone +49-9131-8532352; fax +49-9131-209297;
e-mail berndwr@humgenet.uni-erlangen.de)
Christina Fuchs, Institut rur Humangenetik, Schwabachanlage 10, Erlangen, 91054,
Germany
PROTOCOL