30 Microdissection of Chromosomes and Reverse FISH
393
The volumes (reaction mix A-D) are calculated for four tubes. Two of
them are used for collecting the chromosomal fragments, one serves as
a negative control and the last one as a positive control by adding 5 pg
of genomic DNA.
1. The coverslip is placed across the cut out of the metal plate with the
metaphase spreads facing upwards. The plate is positioned on the microscope working stage with the cut out above the objective.
2. The dissection needle is introduced into the reftllable lead pencil and
attached to the micro-manipulator. Under low magnification (objective lOx) the tip of the needle is centered into the field of vision but
"parked" in a position sufficiently distant above the coverslip.
3. An appropriate metaphase spread is chosen and centered in the field of
vision. The objective is changed to 100 x and the particular chromosome to be dissected is orientated perpendicular to the needle.
4. Now the needle is carefully lowered until the tip becomes visible. Still
above the surface of the coverslip the tip of the needle is positioned
right in front of the chromosomal region to be dissected or the marker
chromosome to be isolated. The needle has to be lowered until it
touches the surface of the coverslip. Any further lowering of the needle
will result in a forward movement of the needle tip and excision of a
chromosomal fragment (Fig. 1).
5. By raising the needle and placing the tip right on the isolated fragment
it usually adheres to the needle and can be taken up.
6. The fragment is transferred to the PCR-tube by breaking off the tip in
the tube.
7. Several fragments are excised in the same manner by repeating step 3
through step 7.
8. Overlay with mineral oil (if a thermocycler without heated lid is used).
Before starting the PCR, a protein digestion is carried out by incubating for
about 60 min at 60°C. This can be done in the thermocycler.
DNA amplification by DOP-PCR
In the original DOP-PCR protocol published by Telenius et al. in 1992, Taq
Polymerase was used for the initial 8 cycles, sometimes referred to as a
393
The volumes (reaction mix A-D) are calculated for four tubes. Two of
them are used for collecting the chromosomal fragments, one serves as
a negative control and the last one as a positive control by adding 5 pg
of genomic DNA.
1. The coverslip is placed across the cut out of the metal plate with the
metaphase spreads facing upwards. The plate is positioned on the microscope working stage with the cut out above the objective.
2. The dissection needle is introduced into the reftllable lead pencil and
attached to the micro-manipulator. Under low magnification (objective lOx) the tip of the needle is centered into the field of vision but
"parked" in a position sufficiently distant above the coverslip.
3. An appropriate metaphase spread is chosen and centered in the field of
vision. The objective is changed to 100 x and the particular chromosome to be dissected is orientated perpendicular to the needle.
4. Now the needle is carefully lowered until the tip becomes visible. Still
above the surface of the coverslip the tip of the needle is positioned
right in front of the chromosomal region to be dissected or the marker
chromosome to be isolated. The needle has to be lowered until it
touches the surface of the coverslip. Any further lowering of the needle
will result in a forward movement of the needle tip and excision of a
chromosomal fragment (Fig. 1).
5. By raising the needle and placing the tip right on the isolated fragment
it usually adheres to the needle and can be taken up.
6. The fragment is transferred to the PCR-tube by breaking off the tip in
the tube.
7. Several fragments are excised in the same manner by repeating step 3
through step 7.
8. Overlay with mineral oil (if a thermocycler without heated lid is used).
Before starting the PCR, a protein digestion is carried out by incubating for
about 60 min at 60°C. This can be done in the thermocycler.
DNA amplification by DOP-PCR
In the original DOP-PCR protocol published by Telenius et al. in 1992, Taq
Polymerase was used for the initial 8 cycles, sometimes referred to as a