Polytene In Situ Hybridization 279
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From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols
Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ
In Situ Hybridization to Polytene Chromosomes
Robert D. C. Saunders
1. Introduction
Since its development by Pardue and Gall (1), the technique of in situ hybridi-
zation to polytene chromosomes has played a central role in the molecular
genetic analysis of Drosophila melanogaster. The power of in situ hybridiza-
tion is largely the result of the scale of polytene chromosomes and, conse-
quently, the high degree of resolution they offer the researcher. The use of
radiolabeled probes has now been largely superseded by faster, nonradioactive
signal detection methods, generally using biotin- or digoxygenin-substituted
probes that also offer greater resolution, because there is less scatter of signal
with immunochemical and immunofluorescent detection than with silver
grains. The utilization of in situ hybridization technology is of particular inter-
est to those engaged in chromosome walking or genome mapping projects, in
which it is essential to check all clones along a chromosome walk by in situ
hybridization in order to identify clones containing repetitive DNA and to avoid
the isolation of clones derived from outside the region of interest. It is also
useful when orienting a chromosome walk and when determining if a particu-
lar clone is derived from DNA uncovered by a deficiency. At least one Droso-
phila genome mapping project (2,3) relied on in situ hybridization to accurately
map sets of overlapping cosmids (contigs) to the polytene chromosome map,
whereas another (4) used in situ hybridization as the sole means of ordering
yeast artificial chromosome (YAC) clones along the genome.
The complete genome sequence of D. melanogaster was published in
2000 (5,6). What role is there for this technique in the postgenomic era?
In fact, there are still situations in which verification of polytene chromo-
some location is important. Examples of such applications are mapping

280 Saunders
chromosome rearrangement breakpoints and work involving related
Drosophila species.
In this chapter, the use of biotin-labeled probes for in situ hybridization to
polytene chromosomes is described.
2. Materials
1. Clean microscope slides (see Note 1).
2. Clean siliconized cover slips, 24-mm square (see Note 1).
3. Clean siliconized cover slips, 22 mm × 50 mm (see Note 1).
4. Compressed air can (e.g., Dust-Off®; Falcon Safety Products, Inc. Sommerville, NJ).
5. 0.7% NaCl.
6. 45% Acetic acid.
7. 1 : 2 : 3 Fixative: 1 vol Lactic acid, 2 vol distilled water, 3 vol glacial acetic acid.
8. Liquid nitrogen.
9. 2X SSC: 300 mM NaCl, 30 mM sodium citrate, pH 7.0.
10. 70 mM Sodium hydroxide, freshly prepared from pellets.
11. 70% and 96% Ethanol.
12. Coplin jars, or similar, for incubating slides.
13. Oligolabeling buffer: Prepare as described in refs. 7 and 8 using the following
solutions:
a. Solution A: Add 9 μL of β-mercaptoethanol, 12.5 μL of 20 mM dATP, 12.5 μL
of 20 mM dCTP, 12.5 μL of 20 mM dGTP to 0.47 mL of solution O.
b. Solution B: 2 M HEPES, pH 6.6.
c. Solution C: Random hexanucleotides (Pharmacia) at a concentration of 90
A260 units/mL.
d. Solution O: 1.25 M Tris-HCl, pH 8.0, 125 mM MgCl2.
Prepare oligolabeling buffer by mixing solutions A, B, and C in the proportions
2 : 5 : 3. Oligolabeling buffer and its constituents should be stored at –20°C.
14. TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.
15. 1 mM Biotin–16-dUTP (Roche). Store at –20°C (see Note 2).
16. 50X Denhardt’s solution: 5 g Ficoll, 5 g polyvinyl pyrrolidone, 5 g bovine serum
albumin (BSA), water to 500 mL. Filter and store at –20°C.
17. 2X Hybridization solution: 8X SSC, 2X Denhardt’s solution, 20% dextran sul-
fate, 0.8% sonicated salmon sperm DNA. Store at –20°C.
18. Plastic box with tightly fitting lid, lined with moist tissue paper.
19. Detek-1 streptavidin–horseradish peroxidase detection kit (Enzo), or ExtrAvidin–
horseradish peroxidase conjugate (Sigma-Aldrich). The Enzo dilution buffer is phos-
phate-buffered saline (PBS) supplemented with 1% BSA, 5 mM EDTA (see Note 2).
20. PBS: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 per 1 L.
21. PBS-TX: PBS containing 0.1% Triton X-100.
22. DAB solution: 0.5 mg/mL Diaminobenzidine in PBS, supplemented with 0.01%
H2O2. DAB is a potent mutagen. Care should be taken at all times when working
with solutions containing DAB. Gloves should be worn and DAB should be dis-