35 PRINS: Hybridization and Primed in Situ Labeling in One Step
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Fig.4. Dideoxy-PRINS with the oligonucleotide (GGTGAG)4GGTG (Tel07, Krejci and Koch
1999) staining a less abundant telomeric repeat located next to the repeat detected in Fig. 2.
In this case ddCTP was used instead of dCTP. The image is produced by combining telomeric signals, in green, with counterstained chromosomes, in red. The target stained by
Telo7 does not light up on all chromosome ends, but when it does light up, it often presents
itself with three or four signals on some chromosome ends, rather than just two
one sequence, and the other strand by another, it is possible to also determine the orientation of the target. It has e.g. been possible to determine
the relative orientation of various telomeric repeat variants with respect to
each other by this method (Krejci and Koch, 1999; Serakinci et aI., 1999).
Materials
10 x dNTP: 1.0 mM each of dATP, dCTP, and dGTP and 0.1 mM
of labeled dUTP in 50% glycerol, prepared from normal nucleotides (Lithium salt, Roche), digoxigenin-dUTP or fluorescein-dUTP
(Roche). For dideoxy-PRINS, the nucleotide not needed for the specific
Oligonucleotide
probes