19 PNA-FISH
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Elegant features ofPNA/DNA duplex formation have made PNA-FISH
more powerful than conventional DNA-FISH. First, the higher affinity of
PNA-DNA duplex and greater accessibility of short and charge-neutral
PNA oligomers (Egholm, et al. 1992, 1993) lead to very strong FISH signals.
Second, these signals are highly saturable, relatively stable, and reproducible. Third, PNA-FISH also shows a very low level of background. Superior hybridization and discrimination ability have brought a potential application in quantitative FISH (Q-FISH, Lansdorp et al. 1996; Slijepcevic et
al. 1997; Zijlmans et al. 1997; Martens, 1998; Hande et al. 1998).
Outline
I.
Chromosome preparations
II.
Post-fIxation in methanol:acetic acid (3:1) for 1-2 h
III.
Dehydration in 70%, 80%, 90%, and 100% ethanol
- Air dry
- lxPBS at 37°C for 5 min
IV.
Denature the chromosomes in 4% formaldehyde at 37°C for
1.5-2 min
V.
Dehydrate slides in the ethanol series and air dry
VI.
Dissolve PNA in a pre-warmed hybridization mix at 37°C for at
least 15 min
VII. Apply 15-25 III of hybridization mix to each slide
VIII. Post-denature chromosomal preps at 80°C for 3 min in a hot plate
IX.
Hybridization at 37°C for 2 h
X.
Post-hybridization washes at RT (room temperature)
(70% formamide 2x15 min and then TNT 3x5 min)
XI.
Slides counterstained with DAPI or PI for subsequent microscope
examination