The basics of locating DNA and RNA

Adapted from the southern blot technique, in situ hybridization for electron microscopy is used to study the location and concentration of nucleic acid sequences (both DNA and messenger RNA) with high precision.

The southern blot technique is performed on gels and is only able to study DNA and RNA at the level of tissues and cells. It can only reveal information about the presence and concentration of such molecules, but not their specific location.

In situ hybridization takes the principles from the southern blot and applied them within cells.

This techniques works around the principle that complementary DNA and RNA sequences will join together, “hybridize” if you will, to form one double stranded molecule.

One of the strands will be naturally within the cell as part of its DNA makeup; we call this the target strand. The other strand is produced synthetically in a lab to be complementary to the target; we call this the probe strand.

During the synthesis of the of the probe strand an antigenic tag (epitope) is attached. This tag has to be a molecule or compound which can be identified using specific antibodies.

It is at this stage that this technique gains a new layer of complexity. Not only does the probe strand have a tag, but the antibody has a tag as well.

Not to worry there are companies nowadays which produce and sell probe strands and the respective antibodies for researchers to use, but I digress.

Back to the tag which is on the antibody, which has bound to the probe strand which has hybridized to the target strand. This tag is an extremely small ball of gold, and is the most crucial piece in the puzzle.

This minuscule ball of gold is what enables us to see the location of the target strand under an electron microscope (image of the electron microscope in the botany department at Melbourne University).

http://www.botany.unimelb.edu.au/botany/em/tem.html

All of the other strands and tags and antibodies turn out completely see-through under an electron microscope, but not gold. Gold is pretty greedy when it comes to free electrons, it will just take and take and take some more. In this way the tiny gold balls appear as black dot under the microscope, and it is these black dots which show us the location of our target strand.

The technique of in situ hybridization for electron microscopy has been used to show; the location of the active DNA within the nucleus, how messenger RNA is trafficked around the cell and where the DNA and RNA from viruses go once they have infected a host cell just to name a few.

Figure 5b from Bassel et al (1994)

I find this multi-layered and intricate technique to be extremely fascinating, however I am not its inventor and so I am more than happy to also inform you about its drawbacks.

This technique can only be used to study DNA and RNA!!!!! In order to do experiments where want to study DNA or RNA along with another molecule such as a protein you would have to perform multiple techniques on the one sample.

The sample preparation time can also be relatively long; especially of you have to do multiple techniques, not to mention that the sample separation is in itself a destructive process. Any trace of life in your sample is pretty quickly removed as the samples needs to be chemically fixed, stained with a toxic heavy metal, completely dehydrated, embedded in resin and finally it needs to be cut into slices no more then 90nM thick, and that is just to prepare the sample for the technique (see page on electron microscopy for the full sample preparation).

Having said this many would argue that the drastic increase in magnification and resolution power of the electron microscope produces results which are unobtainable through other technique and so are worth the time and money.