18 Quantitative DNA Fiber Mapping
243
3. For labeling with either dig-dUTP or FITC-dUTP, add:
- 2.5 III lOx Modified Nucleotide Mixture
- 3.25 III 1 mM dTTP
- 1.751!1 dig-ll-dUTP or FITC-I2-dUTP (1 mM, ROCHE MOLECULAR BIOCHEMICALS, #1093 088)
- 10 1l12.5x Random primers (BioPrime kit, Life Technologies part#
Y01393)
Note: For labeling the DNA with biotin, add 2.5 III 10 x dNTP mix provided with the BioPrime kit (containing biotin-I4-dCTP), 5 III water,
and 10 III 2.5x random primers).
4. Mix well, add 0.5 III DNA polymerase I Klenow fragment (40 units/ill,
Life Technologies, part #YOI396) and incubate in a water bath at 37°C
for 60-120 min.
5. Add 2.5 III of lOx stop buffer (Life Technologies, part #YOII07, part of
the BioPrime kit).
6. Store probe at -20°C until used.
Subprotocol 7
Fluorescence in situ hybridization (FISH)
All hybridizations are carried out overnight at 37°C in a moist chamber.
Fiber hybridizations include a comparatively low concentration of a biotin- or FITC-Iabeled DNA probe prepared from the high molecular weight
DNA that is used to prepare the fibers. This counterstain highlights the
otherwise invisible DNA fibers and allows competitive displacement by
the probes to be mapped along the DNA fiber (Weier et al. 1995, Duell
et aI. 1997). Additionally, one or several cloning vector-specific probes
are included to allow a determination of the orientation of the insert.
Procedure
The hybridization procedure is very similar to protocols used with metaphase spreads:
1. Hybridization mix: combine 1 III of each probe, 1 III of human con
DNA (optional), 1 III of salmon or herring sperm DNA, and 7 III of
hybridization master mix.