19 PNA-FISH
257
- One set of slide racks
- 37
D C water bath
- 37
D C incubator
- PCR with slide holder or heat block
- -20
D C freezer
Procedure
1. Because of the higher Tm of PNA/DNA duplex, shorter 12- to 18-mer
PNAs are widely used in FISH studies. High purine (G > A)-rich sequences are to be avoided owing to difficulty in synthesis. Significant
primer dimers should also be eliminated. A variety of fluorescent dyes
can be labeled at either the 5 or 3 site of PNA molecules. They include
rhodamine, fluorescein, and Texas Red etc. In comparison with fluorescein-labeled PNAs, rhodamine-labeled PNA is dissolved more easily.
A short incubation of the PNA oligomer at 37-50
D C will increase its
solubility.
2. It is recommended that PNA should be lyophilized for shipping and
storage. Because PNA oligomers have an affinity for glass surfaces and
polystyrene, polypropylene or polyethylene materials are recommended for the handing and storage of PNA. Upon arrival from PerSeptive Biosystems, 0.2-2 ml of 0.1 % TFA is added to dissolve PNA in
order to reach an initial PNA concentration at 20 11M. After the PNA is
completely dissolved, it is necessary to re-quantify PNA using a UV
spectrophotometer at 260 nm. PNA can be heated at 50
D C for up to
30 min to improve its solubility. Finally, PNA is aliquoted in 5 or
50 III (equivalent to 0.1 or 1 nmol) volumes, speed-vacuum dried,
and stored at -20
D
e.
3. Metaphase chromosomes or interphase nuclei are made by standard
cytogenetic procedures (Lawrence et al. 1990) and stored at -20
D C if not
being used.
4. We strongly recommend a post-fIxation for all slides. After removal
from the freezer or being freshly made, slides are simply fIxed again
in methanol:acetic acid (2-3:1) for 1-2 h at room temperature, dehydrated in a cold 70, 80, 90, 100% ethanol series, and air dried up to
overnight. If necessary, slides can be treated with pepsin. We found
Proper design and
handling of PNAs
Prepare metaphase
or interphase cells
Post-fix chromosomal preparations
257
- One set of slide racks
- 37
D C water bath
- 37
D C incubator
- PCR with slide holder or heat block
- -20
D C freezer
Procedure
1. Because of the higher Tm of PNA/DNA duplex, shorter 12- to 18-mer
PNAs are widely used in FISH studies. High purine (G > A)-rich sequences are to be avoided owing to difficulty in synthesis. Significant
primer dimers should also be eliminated. A variety of fluorescent dyes
can be labeled at either the 5 or 3 site of PNA molecules. They include
rhodamine, fluorescein, and Texas Red etc. In comparison with fluorescein-labeled PNAs, rhodamine-labeled PNA is dissolved more easily.
A short incubation of the PNA oligomer at 37-50
D C will increase its
solubility.
2. It is recommended that PNA should be lyophilized for shipping and
storage. Because PNA oligomers have an affinity for glass surfaces and
polystyrene, polypropylene or polyethylene materials are recommended for the handing and storage of PNA. Upon arrival from PerSeptive Biosystems, 0.2-2 ml of 0.1 % TFA is added to dissolve PNA in
order to reach an initial PNA concentration at 20 11M. After the PNA is
completely dissolved, it is necessary to re-quantify PNA using a UV
spectrophotometer at 260 nm. PNA can be heated at 50
D C for up to
30 min to improve its solubility. Finally, PNA is aliquoted in 5 or
50 III (equivalent to 0.1 or 1 nmol) volumes, speed-vacuum dried,
and stored at -20
D
e.
3. Metaphase chromosomes or interphase nuclei are made by standard
cytogenetic procedures (Lawrence et al. 1990) and stored at -20
D C if not
being used.
4. We strongly recommend a post-fIxation for all slides. After removal
from the freezer or being freshly made, slides are simply fIxed again
in methanol:acetic acid (2-3:1) for 1-2 h at room temperature, dehydrated in a cold 70, 80, 90, 100% ethanol series, and air dried up to
overnight. If necessary, slides can be treated with pepsin. We found
Proper design and
handling of PNAs
Prepare metaphase
or interphase cells
Post-fix chromosomal preparations