In Situ Hybridization

A modern approach to the specific location of genes on chromosomes is a technique for the hybridization of DNA and RNA “in situ.” With this procedure, specific radioactive RNA or DNA (known as probes) can be isolated (or
synthesized “in vitro”) and then annealed to chromosomes that have been treated in such a manner that their basic double-stranded DNA has been “melted” or dissociated.

In theory, and fortunately in practice, when the DNA is allowed to reanneal, the probe competes for the binding, but only where it mirrors a complementary sequence. Thus, RNA will attach to the location on the chromosome where the code for its production is to be found. DNA will anneal to either RNA that is still attached to a chromosome, or to the complementary sequence DNA strand within the chromosome. Since the probe is radioactive, it can be localized via autoradiographic techniques.

Finally, it is possible to produce an RNA probe that is synthesized directly from repetitive sequences of DNA, such as that found within the nucleolar organizer region of the genome. This RNA is known as cRNA (for copied RNA)
and is a convenient source of a probe for localizing the nucleolar organizer gene within the nucleus, or on a specific chromosome.

The use of in situ hybridization begins with good cytological preparations of the cells to be studied, and thepreparation of pure radioactive probes for the analysis. The details depend upon whether the hybridization is between DNA (probe) and DNA (chromosome), DNA (probe) and RNA (chromosome), or between RNA (probe) and DNA (chromosome).


Preparation of the Probe
Produce radioactive RNA by incubating the cells to be measured in the presence of 3H-uracil, a specific precursor to RNA. Subsequent to this incubation, extract rRNA from the sample and purify through differential centrifugation, column chromatography or electrophoresis. Dissolve the radioactive RNA probe in 4X saline-citrate containing 50% formamide to yield a sample that has 50000 to 100000 counts per minute, per 30 microliter sample, as determined with a scintillation counter. Add the formamide to prevent the aggregation of RNA.


Preparation of the Slides
Fix the materials to be studied in either 95% ethanol or in 3:1 methanol:water, attach to presubbed slides (as squashes for chromosomes) and air dry.

Hybridization
Place the air-dried slides into a moist chamber, usually a disposable petri dish containing filter paper, and carefully place 30 microliters of RNA probe in 4X SSC-50% formamide onto the sample. Carefully add a cover slip (as in the preparation of a wet mount), place the top on the container and place in an incubator at 37°C for 6–12 hours.

Washing

1. Pick up the slides and dip into 2X SSC so that the coverglass falls off.
2. Place the slides in a coplin jar containing 2X SSC for 15 minutes at room temperature.
3. Transfer the slides to a treatment with RNase (50 microgram/mL RNase A, 100 units/mL RNase T1 in 2X SSC) at 37°C for 1 hour.
4. Wash twice in 2X SSC, 15 minutes each.
5. Wash twice in 70% ethanol, twice in 95% ethanol, and air dry.

Autoradiography
Add photographic emulsions to the slides and after a suitable exposure period, develop the slides, counterstain, and add cover slips.
Analyze the slides by determining the location of the radioactive probe on the chromosomes or within the nuclei.