88
THOMAS LIEHR
Comments
1. RNase and pepsin pretreatment conditions should be tested in each
laboratory on a single slide first. Both RNase and pepsin concentrations can be too stringent, resulting in clean slides without any remaining nuclei.
2. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. Iflonger storage is necessary slides are stable at -20°C for
several months.
3. In other FISH-protocols with denaturation times of2-3 min only, the
maintenance of available metaphase chromosomes is the main aspect,
which is of no significance in the actual protocol.
4. Incubation can be stopped - if necessary - after 48 or 96 h, as well.
While in the first case weaker signals are possible, in the second
case some cross-hybridization problems may arise.
5. During the washing steps it is important to prevent the slide surfaces
drying out, otherwise background problems may arise.
Acknowledgements. Supported by the Wilhelm Sander-Stiftung (99.105.1) and the EU
(QLRT-1999-31590). The continuos support of the Carl Zeiss GmbH (Jena, Germany) is
gratefully acknowledged.
References
Langer, PR, Waldrop, AA, Ward, DC (198l) Enzymatic synthesis of biotin-labeled
polynucleotides: Novel nucleic acid affinity probes. Proc Nat! Acad Sci USA,
78:6633-6637
Liehr, T, Thoma, K, Kammler, K, Gehring, C, Ekici, A, Bathke, KD, Grehl, H, Rautenstrauss, B (1995) Direct preparation of uncultured EDTA-treated or heparinized
blood for interphase FISH analysis. Appl Cytogenet, 21:185-188
Liehr, T, GreW, H, Rautenstrauss, B (1997) Molecular diagnosis of PMP22-associated
neuropathies using fluorescence in situ hybridization (FISH) on archival peripheral
nerve tissue preparations. Acta Neuropathol, 94:266-271
Patel, PI, Lupski, JR (1994) Charcot-Marie-Tooth disease: a new paradigm for the mechanism of inherited disease. Trends in Genetics, 10:128-133
Pinkel, D, Straume, T, Gray, JW (1986) Cytogenetic analysis using quantitative, high
sensitivity, fluorescence hybridization. Proc Nat! Acad Sci USA, 83:2934-2938
Sawyer, JR, Johnson, MP, Miller, OJ (1992) Traditional and molecular cytogenetics.
J Reprod Med 37, 485-498.
THOMAS LIEHR
Comments
1. RNase and pepsin pretreatment conditions should be tested in each
laboratory on a single slide first. Both RNase and pepsin concentrations can be too stringent, resulting in clean slides without any remaining nuclei.
2. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. Iflonger storage is necessary slides are stable at -20°C for
several months.
3. In other FISH-protocols with denaturation times of2-3 min only, the
maintenance of available metaphase chromosomes is the main aspect,
which is of no significance in the actual protocol.
4. Incubation can be stopped - if necessary - after 48 or 96 h, as well.
While in the first case weaker signals are possible, in the second
case some cross-hybridization problems may arise.
5. During the washing steps it is important to prevent the slide surfaces
drying out, otherwise background problems may arise.
Acknowledgements. Supported by the Wilhelm Sander-Stiftung (99.105.1) and the EU
(QLRT-1999-31590). The continuos support of the Carl Zeiss GmbH (Jena, Germany) is
gratefully acknowledged.
References
Langer, PR, Waldrop, AA, Ward, DC (198l) Enzymatic synthesis of biotin-labeled
polynucleotides: Novel nucleic acid affinity probes. Proc Nat! Acad Sci USA,
78:6633-6637
Liehr, T, Thoma, K, Kammler, K, Gehring, C, Ekici, A, Bathke, KD, Grehl, H, Rautenstrauss, B (1995) Direct preparation of uncultured EDTA-treated or heparinized
blood for interphase FISH analysis. Appl Cytogenet, 21:185-188
Liehr, T, GreW, H, Rautenstrauss, B (1997) Molecular diagnosis of PMP22-associated
neuropathies using fluorescence in situ hybridization (FISH) on archival peripheral
nerve tissue preparations. Acta Neuropathol, 94:266-271
Patel, PI, Lupski, JR (1994) Charcot-Marie-Tooth disease: a new paradigm for the mechanism of inherited disease. Trends in Genetics, 10:128-133
Pinkel, D, Straume, T, Gray, JW (1986) Cytogenetic analysis using quantitative, high
sensitivity, fluorescence hybridization. Proc Nat! Acad Sci USA, 83:2934-2938
Sawyer, JR, Johnson, MP, Miller, OJ (1992) Traditional and molecular cytogenetics.
J Reprod Med 37, 485-498.