John M. S. Bartlett.pdf - Bio-Nica.info

Cycling Primed In Situ Amplification 431
9. Propidium iodide binds specifically to DNA and emits a red fluorescence distinct from
FITC. If desired, DAPI (4′,6-Diamidino-2-phenylindole 2 HCl; Sigma) can also be used
at the same concentration. This emits a strong blue fluorescence, which sometimes aids in
locating the cells on the slide. As an alternative detection system, Texas Red-conjugated
reagents are available for detection of DIG or biotin. This can be combined with the DAPI
counterstain, leaving the green channel free for detection of a third label, for example,
via immunostaining (7,9).
References
1. Gosden, J. and Lawson, D. (1994) Rapid chromosome identification by oligonucleotideprimed
in situ synthesis (PRINS). Hum. Mol. Genet. 3, 931–936.
2. Hindkjaer, J., Koch, J., Terkelsen, C., Brandt, C. A., Kolvraa, S., and Bolund, S. (1994)
Fast, sensitive multicolour detection of nucleic acids in situ by primed in situ labelling
(PRINS). Cytogenet. Cell Genet. 66, 152–154.
3. Trask, B. J. (1991) Fluorescence in situ hybridization: Applications in cytogenetics and
gene mapping. Trends Genet. 7, 149–54.
4. Koch, J., Hindkjaer, J., Kolvraa, S., and Bolund, L. (1995) Construction of a panel of
chromosome-specific oligonucleotide probes (PRINS-primers) useful for the identification
of individual human chromosomes in situ. Cytogenet. Cell Genet. 71, 142–147.
5. Gosden, J. and Hanratty, D. (1993) PCR in situ: A rapid alternative to in situ hybridisation
for mapping short, low-copy number sequences without isotopes. BioTechniques 15,
78–83.
6. Spowart, G. (1994) Mitotic metaphase chromosome preparation from peripheral blood for
high resolution, in Methods in Molecular Biology, Vol. 29: Chromosome Analysis Protocols
(Gosden, J. R., ed.), Humana, Totowa, NJ, pp. 1–10.
7. Paskins, L., Brownie, J., and Bull, J. (1999) In situ polymerase chain reaction and cycling
primed in situ amplification: improvements and adaptations. Histochem. Cell Biol. 111,
411– 416.
8. Jackson, P. and Blyth, D. (1993) Immunolabelling techniques for light microscopy,
in Immuncytochemistry, a Practical Approach (Beesley, J. E., ed.), IRL, Oxford, UK.
pp. 15– 42.
9. Speel, J. M., Lawson, D., Ramaekers, C. S., Gosden, J. R., and Hopman, A. H. N. (1997)
Combined immunocytochemistry and PRINS DNA synthesis for simultaneous detection
of phenotypic and genomic parameters in cells, in Methods in Molecular Biology, Vol. 71:
PRINS and in Situ PCR Protocols (Gosden, J.R., ed.), Humana, Totowa, NJ, pp. 53–60.