In Situ Hybridization, Fluorescence And Fish Fluorescent

What Is In Situ Hybridization?

In situ hybridization is the technique by which a labeled single-stranded DNA or RNA molecule in solution is hybridized with immobilized single-stranded RNA or DNA in a tissue or tissue section. And by having a label on the molecule in solution, eventually, the hybrid is visible in the tissue section as seen through a microscope.

 In Situ Hybridization Fluorescence

This in situ hybridization technique was carried out successfully for the first time in 1968. The origin of in situ hybridization, at least as an intellectual phenomenon, stems from experiments that had been carried out much earlier, mainly around 1950, by two researchers named Coons and Kaplan. . What Coons and Kaplan had done was to develop the technique of immunocytochemistry, a technique by which a fluorescent antibody was made to bind to the immobilized antigen in a section of tissue so that the position of specific protein molecules in the cells could be detected. cells by their fluorescence.

Coons And Kaplan In Situ Hybridization Technique

Using the Coons and Kaplan in situ hybridization technique. specific protein molecules in tissues and it occurred to me that it would be wonderful if specific nucleic acids could be detected in tissues. Actually, he was thinking less about nucleic acids than genes because he had been studying the giant chromosomes of salamanders.

The giant chromosomes of Drosophila, in these chromosomes, you can see bands that were considered as genes by the people who worked with them at that time. Now we know that this is a bit more complicated than that, but it seemed that we had here a tissue, a chromosome where specific genes, specific nucleic acids.

It should be possible to do something comparable to immunofluorescent staining to detect specific genes in these tissues. It became possible to think of doing this kind of thing in the early 1960s when researchers discovered that you could take double-stranded DNA, denature it into its single strands, and immobilize those strands on a nitrocellulose filter.

In Situ Hybridization RNA

This was done by a couple of researchers named Gillespie and Spiegelman, and what they showed was that by having denatured DNA on a filter, one could hybridize radioactive RNA to it and actually determine, quantitatively, the amount of a particular genetic sequence in it. filtering. Well, if they can do it with a filter, you should be able to do it with a slide. " So, I tried to hybridize a radioactive RNA with it but got no signal.

In this case, look for radioactivity by autoradiography, that is, covering the tissue

with a thin layer of photographic emulsion and looking for silver grains in the emulsion caused by radioactivity from the probe. Like I said, nothing happened, and I more or less abandoned that technique. That was from the early to mid-1960s. Now, there was a break, quite accidentally, from the studies that I and others had been doing on salamander oocytes and frog oocytes, and what we found was that in the nucleus of the giant oocyte, derived from this large oocyte. The oocyte is the frog egg inside this giant nucleus are of course the chromosomes, but in addition, we found that there were several hundred or thousands of nucleoli, these round bodies that are shown here on this particular slide.

Now the interesting thing about these nucleoli that we discovered was that

contained DNA and were specifically the genes encoding ribosomal RNA

that were in these nucleoli. This was a very unusual situation to have DNA off chromosomes, but it was quite clear from the work that had been done in my lab,

as well as in the laboratory of Don Brown and Oscar Miller, that there was extrachromosomal DNA outside in these nucleoli.

The origin of that DNA and what I found was that it was actually synthesized at a specific time in the early history of oocyte formation, and you could see in these early nuclei of early cells that there was a layer of DNA. In these two nuclei, you have the chromosomes, but then you have this huge layer of extra DNA that was actually the ribosomal genes. Somehow they come off the chromosome and amplify themselves to give you this specific sequence, a lot of this specific sequence.

So it occurred to me that if there was any possibility of doing an in situ hybridization, this was the case that it would work. So I took a radioactive ribosomal RNA and hybridized it with cells of this type, and I was very pleased to see in the first experiments that we got good in situ hybridization. This cell is black due to the silver grains in the photographic emulsion that have been exposed by radioactivity.

of the hybridization probe.

In Situ Hybridization Radioactive

So this is a case where we have hybridized radioactive RNA - ribosomal RNA with ribosomal DNA in the cell. So that immediately led to a post with my student Mary Lou Pardue, who joined me on this work, and this was the first successful in situ hybridization. Now, we wanted to test it on some other tissue or DNA, but there were very few examples because at that time it was not possible to isolate specific DNA or RNA sequences. What allowed us to see another situation came from studies that had been carried out with mouse DNA, and the researchers had found that when mouse DNA was spun on a cesium chloride (CsCl) gradient, it separated into two fractions. , the main peak and a satellite peak called a satellite simply because it had a slightly different density and was separated from the bulk of the DNA.

We took this satellite DNA, made it radioactive, and hybridized it to mouse chromosomes, not knowing at all if it would hybridize, if it was even on the chromosomes, it could have been a virus or some other infectious particle, but we were very pleased to see that the DNA The mouse satellite specifically hybridized to a portion of each chromosome, each of the chromosomes having a bit of radioactivity at the end of the chromosome.

This region of the chromosome was known as heterochromatin and was known cytologically as a specific region of the chromosome for several years. This immediately led us to think that we should be able to try the same technique and get similar results when looking at Drosophila chromosomes, and Drosophila chromosomes were especially interesting because it was known from genetics that all genes were in the so-called euchromatic regions. of the chromosome.

In Situ Hybridization DNA

So it occurred to us that Drosophila must have a lot of DNA similar to mouse satellite DNA. So we centrifuged that DNA in a cesium chloride gradient and found, in fact, that Drosophila had a lot of satellite DNA. This species of Drosophila virilis

The one we were working on had three huge satellite spikes.

In addition to the main DNA spike, we took the satellite DNA, made it radioactive, hybridized it to chromosomes, and indeed it hybridized to the heterochromatic regions. And this was particularly interesting because it showed that the heterochromatic regions, which we knew from genetics to have no genes, consisted of this satellite-like DNA.

That our subsequent studies showed that it consisted of very simple sequences, that they were either incapable of coding for genes, or they were incapable of being genes and coding for proteins. So the in situ hybridization technique at this point was practically limited to sequences that were present in large numbers in cells.

In Situ Hybridization FISH

Eventually, it became possible, by having increasingly sensitive probes, in particular, later techniques were developed to make fluorescent probes for in situ hybridization. thus, in situ hybridization, ISH or "ish", became fluorescent in situ hybridization, or "fish", FISH, and the techniques have become increasingly sensitive, to the point that, today, individual DNA and RNA molecules can be detected in tissue sections.