Signal Detection

1. Take the slides from the final wash and pass them through two 5-min washes in PBS.

2. Wash the slides for 2 min in PBS-TX

3. Rinse in PBS. Do not allow the slides to dry out during signal detection.

4. Make a 1 : 250 dilution of streptavidin-horseradish peroxidase conjugate in the buffer supplied with the Enzo kit. Alternatively, ExtrAvidin-horseradish peroxi-dase conjugate (Sigma) can be used, at the same dilution. Apply 50 |L to the chromosomes and cover with a 22 x 50 mm2 cover slip. Replace the slides in the humid box and incubate at 37°C for 30 min.

5. Wash off unbound streptavidin conjugate by passing the slides through the PBS and PBS-TX washes described in steps 1-3. Drain the slides, but do not allow them to dry.

6. Place 50 ||L of DAB solution onto the chromosomes and cover with a 22 x 50-mm2 cover slip. This solution should be made up fresh because hydrogen peroxide decays rapidly. Take care when working with DAB, as it is a potent carcinogen. Follow the guidelines for use and disposal described in Subheading 2., item 22.

7. Incubate at room temperature for 10-15 min in the humid box. Rinse the slides in PBS and examine under phase contrast. The signal appears blackish-brown, sometimes quite refractile in strong cases. If the signal seems weak, add more DAB solution and incubate longer. If the signal is strong enough, rinse the slide well with distilled water (see Note 7).

8. Stain in Giemsa's stain for 1 min, rinsing off excess stain in running water for a few seconds. Allow the slides to air-dry. Check that the staining is sufficiently intense. Overstained chromosomes can be destained in 10 mM sodium phosphate buffer, pH 6.8, and understained chromosomes can be restained. The preparation should be mounted under a siliconized cover slip with DPX mounting medium. DPX is a xylene-soluble mountant, which does not affect either the Giemsa stain or DAB deposit, and the slides should last for many years. It is convenient to seal the edges of the cover slip with nail polish to prevent immersion oil from seeping under the cover slip.

The preparations should be examined under phase contrast. Some results are shown in Fig. 1, although photographic reproduction is most successful with color film.

4. Notes

1. Make sure the slides and cover slips are scrupulously clean, especially of lint from any tissue paper used to clean them; thus, only lint-free tissue paper should be used. It is good practice to clean slides and cover slips immediately prior to use by dipping in ethanol and wiping. Excess ethanol and lint can be removed by using a photographer's compressed air can (e.g., Dust-Off).

2. The biotin-labeling methods of probe preparation are not the only methods available to the researcher. Other labeling systems include the digoxygenin system (Roche), and alternatives to random priming for incorporation of label, such as photobiotin labeling, can be used. When selecting a detection system for an experiment, several factors must be kept in mind. First, the durability of the specimen is important. Signals visualized with horseradish peroxidase and DAB offer the advantage of long-term stability, compared with the fluorescent methods, an important feature when engaged in long-range chromosome walking or genome mapping. Second, one should assess the degree of sensitivity required for a given experiment. For example, methods for directly labeling probe DNA with fluorescent dyes, although quicker, do not offer the same degree of sensitivity as the two-step detection systems. Third, when choosing a method that employs an enzymatic reaction for detection, such as alkaline phosphatase or horseradish peroxidase, care should be taken to select a substrate whose reaction product is both stable and insoluble in the mountant in use and that contrasts well with the chromosome counterstain.

Fig. 1. In situ hybridization of biotin-labeled probe DNA to D. melanogaster polytene chromosomes. The signals have been detected using streptavidin-horseradish peroxidase conjugate, with DAB as the substrate, and the chromosomes are counter-stained with Giemsa. (A) Mapping a cosmid clone, using a 2.8-kb restriction fragment as a probe. The signal is indicated by the arrow and lies in bands 96A21-25. (B) Mapping a clone relative to a chromosome rearrangement. The probe is a cosmid/phage containing an insert derived from bands 96B1-10, hybridized to T(Y;3)B197/+ chromosomes. The signal lies on chromosome 3, in bands 96B1-10, proximal to the breakpoint. (C) Hybridization of PCR amplified DNA derived from microdissection of division 1 (9). (D) Hybridization of PCR amplified DNA derived from microdissection of subdivision 25A (9). Scale bar: 20 |im.

Fig. 1. In situ hybridization of biotin-labeled probe DNA to D. melanogaster polytene chromosomes. The signals have been detected using streptavidin-horseradish peroxidase conjugate, with DAB as the substrate, and the chromosomes are counter-stained with Giemsa. (A) Mapping a cosmid clone, using a 2.8-kb restriction fragment as a probe. The signal is indicated by the arrow and lies in bands 96A21-25. (B) Mapping a clone relative to a chromosome rearrangement. The probe is a cosmid/phage containing an insert derived from bands 96B1-10, hybridized to T(Y;3)B197/+ chromosomes. The signal lies on chromosome 3, in bands 96B1-10, proximal to the breakpoint. (C) Hybridization of PCR amplified DNA derived from microdissection of division 1 (9). (D) Hybridization of PCR amplified DNA derived from microdissection of subdivision 25A (9). Scale bar: 20 |im.

3. Polytene chromosome maps are available for many Drosophila species. Sorsa (10) has compiled a list of all maps of drosophilid polytene chromosomes. For D. melanogaster, the 1935 Bridges map (11) and the Lefevre (12) photomap are indispensable. These are available from Academic Press, in a folder together with the Bridges' revised maps (13-15).

4. One of the most important factors in successful in situ hybridization experiments is the quality of the polytene chromosomes. There are many ways in which polytene chromosomes can be prepared, differing mostly in the manner by which the chromosomes are spread and squashed. Allowing the cover slip to slip sideways when spreading causes the chromosome arms to stretch. Overstretched chromosomes can make analysis of the in situ hybridization difficult. Poor chromosome morphology can also result from denaturing chromosomes for too long in alkali and from other, poorly understood, fixation problems. If the chromosome morphology is repeatedly found to be puffy and swollen, try the alternative denaturation method of boiling, which often preserves the morphology better than alkali denaturation.

5. The presence of repetitive DNA within a cloned segment of DNA can prevent easy determination of the chromosomal site of origin of the clone. The use of sibling species can resolve this problem. For example, D. simulans and D. mauritiana polytene chromosomes have been used (2) in mapping cosmids containing cloned segments of D. melanogaster DNA. This is possible because the sibling species have different amounts of repetitive DNA and different populations of transposable elements.

6. High background on preparations is generally associated with poor incorporation of a biotinylated nucleotide and inefficient removal of unincorporated nucleotides prior to hybridization.

7. If no signal is seen when using a biotinylated probe, test the probe as follows. Make a dot blot with a series of dilutions of unlabeled probe DNA and a series of dilutions of probe DNA. Hybridization under standard filter hybridization conditions followed by signal detection using the same system as used for in situ hybridization will indicate whether the problem lies in probe preparation or in signal detection. A systemic problem where no signals are obtained with a variety of probes may indicate that the working DAB solution has decayed. Generally, this can be rectified by using fresh hydrogen peroxide. Stocks of hydrogen peroxide should be replaced regularly.

References

1. Pardue, M.-L., Gerbi, S.A., Eckhardt, R. A., and Gall, J. G. (1969) Cytological localization of DNA complementary to ribosomal RNA in polytene chromosomes of Diptera. Chromosoma 29, 268-290.

2. Siden-Kiamos, I., Saunders, R. D. C., Spanos, L., et al. (1990) Towards a physical map of the Drosophila melanogaster genome: mapping of cosmid clones within defined genomic divisions. Nucleic Acids Res. 18, 6261-6270.

3. Kafatos, F. C., Louis, C., Savakis, C., et al. (1991) Integrated maps of the Drosophila genome: progress and prospects. Trends Genet. 7, 155-161.

4. Ajioka, J. W., Smoller, D. A., Jones, R. W., et al. (1991) Drosophila genome project: one-hit coverage in yeast artificial chromosomes. Chromosoma 100, 495-509.

5. Adams, M. D., Celniker, S. E., Holt, R. A., et al. (2000) The genome sequence of Drosophila melanogaster. Science 287, 2185-2195.

6. Benos, P. V., Gatt, M. L., Ashburner, M., et al. (2000) From sequence to chromosome: the tip of the X chromosome of D. melanogaster. Science 287, 2220-2222.

7. Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13.

8. Feinberg, A. P. and Vogelstein, B. (1984) Addendum to Feinberg and Vogelstein (1983). Anal. Biochem. 137, 266-267.

9. Saunders, R. D.C., Glover, D. M., Ashburner, M., et al. (1989) PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning. Nucleic Acids Res. 19, 9027-9037.

10. Sorsa, V. (1988) Chromosome maps of Drosophila, CRC Press, Boca Raton, FL.

11. Bridges, C. B. (1935) Salivary chromosomes. With a key to the banding of the chromosomes of Drosophila melanogaster. J. Heredity 26, 60-64.

12. Lefevre, G. (1976) A photographic representation and interpretation of the poly-tene chromosomes of Drosophila melanogaster salivary glands, in The Genetics and Biology of Drosophila, (Ashburner, M. and Novitski, E., eds), Academic, New York, Vol. 1a, pp. 31-66.

13. Bridges, C. B. (1938) A revised map of the salivary gland X-chromosome. J. Heredity 29, 11-13.

14. Bridges, C. B. and Bridges, P. N. (1939) A new map of the second chromosome: a revised map of the right limb of the second chromosome of Drosophila melanogaster. J. Heredity 30, 475-476.

15. Bridges, P. N. (1941) A revision of the salivary gland 3R-chromosome map of

Drosophila melanogaster. J. Heredity 32, 299-300.

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