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

462 Speel, Ramaekers, and Hopman
buffer (500 mM Tris-HCl, pH 7.2; 100 mM MgSO 4 ; 100 mM DTT; 1.5 mg.mLBSA), 1 U
Klenow DNA polymerase (Roche), and distilled water to 50 µL. Incubate slide with
40 µL under a coverslip for 1 h at 37°C in a humid chamber, followed by dehydration and
airdrying before running the next PRINS reaction (5).
13. Amplification of PRINS signals can be achieved as follows:
a. AvFITC detection of Biotin-16-dUTP may be followed by incubation with biotinylated
goat anti-avidin (Vector), 1100 diluted in blocking buffer, and again AvFITC.
b. SHADigFITC detection of digoxigenin-11-dUTP may be followed by incubation
with FITC-conjugated anti-sheep IgG (Roche), or as described for FITC-12-dUTP
amplification.
c. Fluorescein-12-dUTP signals may be amplified by incubation with monoclonal mouse
or polyclonal rabbit anti-FITC (Dako) and FITC-conjugated rabbit anti-mouse IgG or
swine anti-rabbit IgG (Dako).
d. Amplification of PRINS signals may also be achieved by using peroxidase-mediated
deposition of hapten- or fluorochrome-labeled tyramides (28–30).
14. The time required will vary according to the repeated DNA family of sequences under study
(size of target) and the cell type from which the chromosome preparations are made.
15. This again may vary according to the cell type and target size under study.
16. The slides can be stored in the dark for several weeks at room temperature at this stage.
References
1. Koch, J., Kölvraa, S., Petersen, K. B., Gregersen, N., and Bolund, L. (1989) Oligonucleotidepriming
methods for the chromosome-specific labelling of alpha satellite DNA in situ.
Chromosoma 98, 259–265.
2. Koch, J., Mogensen, J., Pedersen, S., Fischer, H., Hindkjder, J., Kölvraa, S., et al. (1992)
Fast one-step procedure for the detection of nucleic acids in situ by primer-induced
sequence-specific labeling with fluorescein-12-dUTP. Cytogenet. Cell. Genet. 60, 1–3.
3. Gosden, J., Hanratty, D., Starling, J., Fantes, J., Mitchell, A., and Porteous, D. (1991)
Oligonucleotide-primed in situ DNA synthesis (PRINS): A method for chromosome
mapping, banding, and investigation of sequence organization. Cytogenet. Cell. Genet.
57, 100–104.
4. Gosden, J. and Lawson, D. (1994) Rapid chromosome identification by oligonucleotideprimed
in situ DNA synthesis (PRINS). Hum. Mol. Genet. 3, 931–936.
5. Speel, E. J. M., Lawson, D., Hopman, A. H. N., and Gosden, J. (1995) Multi-PRINS:
multiple sequential oligonucleotide primed in situ DNA synthesis reactions label specific
chromosomes and produce bands. Hum. Genet. 95, 29–33.
6. Speel, E. J. M., Lawson, D., Ramaekers, F. C. S., Gosden, J. R., and Hopman, A. H. N.
(1996) Rapid brightfield detection of oligonucleotide primed in situ (PRINS)-labeled DNA
in chromosome preparations and frozen tissue sections. BioTechniques 20, 226–234.
7. Gosden, J. R., ed. (1997) PRINS and In Situ PCR Protocols. Methods in Molecular Biology,
Vol. 71, Humana Press, Totowa, NJ.
8. Wilkens, L., Tchinda, J., Komminoth, P., and Werner, M. (1997) Single- and double-color
oligonucleotide primed in situ labeling (PRINS): Applications in pathology. Histochem.
Cell Biol. 108, 439– 446.
9. Hindkjder, J., Koch, J., Terkelsen, C., Brandt, C. A., Kölvraa, S., and Bolund, L. (1994)
Fast, sensitive multicolor detection of nucleic acids in situ by primed in situ labeling
(PRINS). Cytogenet. Cell. Genet. 66, 152–154.
10. Speel, E. J. M. (1999) Detection and amplification systems for sensitive, multiple-target
DNA and RNA in situ hybridization: Looking inside cells with a spectrum of colors.
Histochem. Cell Biol. 112, 89–113.