35 PRINS: Hybridization and Primed in Situ Labeling in One Step
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To cover a smaller area, prepare less reaction mixture (and use a
smaller coverslip). The amount (in Ill) of mixture needed is roughly
proportional to the length of the coverslip (in mm).
2. Denaturation ofthe chromosomal DNA: As soon as the polymerase has
been added to the reaction mixture, it should be applied to a freshly
prepared slide and spread with a coverslip of a suitable size (25 x 50 mm
to cover a full slide). Denature the chromosomal DNA by placing the
slide for 4 min on a 94°C hot plate covered by a lid.
3. Probe annealing and chain elongation: Transfer the slide quickly to 5565°C and incubate for 5-60 min (depending on the probe). If a specialized incubator is used, the machine "handles" steps 2 and 3, so just
spread the reaction mixture with the coverslip, put the slide in the machine, press the button to start the reaction and transfer the slide to
stop buffer when the program is completed.
4. Place the slide in stop buffer preheated to the temperature used in
step 3, for 1 min to terminate PRINS labeling.
5. Transfer the slide to 50 ml of washing buffer and wash for 3 min at
room temperature.
6a. If fluorochrome labeled nucleotides have been used, the slide can now
be counterstained, mounted and evaluated under the microscope.
6b. If digoxigenin labeled nucleotides have been used, they need to be
visualized with anti-digoxigenin antibody as described below.
Dideoxy-PRINS
Perform the PRINS procedure exactly as above, but replace the nUcleotide(s) not needed for DNA synthesis from correctly hybridized probes
with the corresponding dideoxynucleotides (e.g. replace dGTP when
synthesizing the CCCTAA element of the human telomere).
Multicolor PRINS and PRINS-FISH
1. Perform the PRINS procedure from above using, for instance, digoxigenin-ll-dUTP (Roche) as the labeled nucleotide.
469
To cover a smaller area, prepare less reaction mixture (and use a
smaller coverslip). The amount (in Ill) of mixture needed is roughly
proportional to the length of the coverslip (in mm).
2. Denaturation ofthe chromosomal DNA: As soon as the polymerase has
been added to the reaction mixture, it should be applied to a freshly
prepared slide and spread with a coverslip of a suitable size (25 x 50 mm
to cover a full slide). Denature the chromosomal DNA by placing the
slide for 4 min on a 94°C hot plate covered by a lid.
3. Probe annealing and chain elongation: Transfer the slide quickly to 5565°C and incubate for 5-60 min (depending on the probe). If a specialized incubator is used, the machine "handles" steps 2 and 3, so just
spread the reaction mixture with the coverslip, put the slide in the machine, press the button to start the reaction and transfer the slide to
stop buffer when the program is completed.
4. Place the slide in stop buffer preheated to the temperature used in
step 3, for 1 min to terminate PRINS labeling.
5. Transfer the slide to 50 ml of washing buffer and wash for 3 min at
room temperature.
6a. If fluorochrome labeled nucleotides have been used, the slide can now
be counterstained, mounted and evaluated under the microscope.
6b. If digoxigenin labeled nucleotides have been used, they need to be
visualized with anti-digoxigenin antibody as described below.
Dideoxy-PRINS
Perform the PRINS procedure exactly as above, but replace the nUcleotide(s) not needed for DNA synthesis from correctly hybridized probes
with the corresponding dideoxynucleotides (e.g. replace dGTP when
synthesizing the CCCTAA element of the human telomere).
Multicolor PRINS and PRINS-FISH
1. Perform the PRINS procedure from above using, for instance, digoxigenin-ll-dUTP (Roche) as the labeled nucleotide.