Fluorescent in situ hybridisation (FISH)

 Fluorescent in situ hybridisation, or FISH is a technique that uses molecular DNA probes to detect complementary sequences along a chromosome. Unlike whole genome analyses, such as karyotyping or array CGH, FISH uses DNA-specific probes and will provide information both about the copy number of a chromosome region-- so that's whether a region's deleted or duplicated-- but also about the locational position of that region.

 Let's spend a bit of time thinking about how FISH works. A FISH probe is designed which is complementary to the strand of DNA which you want to identify. The probe is labelled or tagged with a coloured fluorochrome. The chromosomes of the target cells are arrested in metaphase. 

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The cells in the chromosomes are fixed onto the surface of a glass slide. The slide is treated so that the genomic DNA is denatured into single strands. The slide is then washed with the single strand probe DNA. And the probe anneals, or hybridises, to the single-stranded target DNA, which is still in its natural position on the chromosome. That's a really key feature of FISH. So it is looking at the target DNA in situ. And because the probe is being tagged with a fluorochrome, which is a molecule which fluoresces when it is excited by a particular wavelength of light, it can be visualised using a fluorescent microscope.

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 Indeed, several different probes can be hybridised, observed, and then compared simultaneously by using different colours and combinations of these fluorochromes for each probe.

There are four major groups of FISH probes, which anneal to different types of DNA. Alpha satellite probes are complementary to the repetitive DNA sequences found at the centromere. Although there is considerable overlap between the DNA content at the centromere, there's also usually enough differences between chromosomes that individual chromosomes can be distinguished. However, that's not true for chromosomes 13 and 21. And it's also very difficult to distinguish chromosome 14 from chromosome 22 with an alpha satellite probe.

And as is often the case, it's actually usually really important to distinguish chromosomes 13 and 21, for instance, if a trisomy is suspected prenatally. And so in this situation, it's important to use other more specific probes that are specific to that chromosome region. Beta satellite probes are designed to anneal to the non-transcribed sequences of the acrocentric chromosomes. And these are chromosomes 13, 14, 15, 21, and 22. Beta satellite probes might be used to interrogate, for instance, extra material on the short arm of an acrocentric chromosome. Unique sequence probes are designed to anneal to specific chromosome regions. And these probes identify sequences usually present in only one copy number per chromosome.

Unique sequence probes are therefore locus specific and can detect a specific chromosome region or gene. And then finally, whole chromosome paints consist of a number of probes, which cover the whole of the chromosome other than the centromere region and contain a mixture of overlapping probes. 

And this is a picture of a whole chromosome paint. And you'll see that it will paint the entire length of a chromosome, and can be used to identify the origin of unidentified material or rearranged chromosomes or marker chromosomes. 

So let's think a bit about the applications of FISH. Historically, FISH was used in a wide variety of applications, many of which are listed here on this slide. However, the technique is time-consuming, costly, and laborious.

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In many areas, it's now been superseded by newer, faster techniques. For instance, prenatal aneuploidy screening is now commonly done using QF-PCR. And array CGH has superseded FISH as the go-to test for microdeletion and telomere screening. Nevertheless, FISH still has a role in confirming array CGH findings. And it can be used to clarify the nature of a balanced or unbalanced translocation, and has found an increasing variety of applications in hemato-oncology. 

Let's have a quick look at how FISH was used for aneuploidy screening. So I think this nicely shows the visual nature of FISH. The image here shows a slide with trisomy 21. You'll see that two chromosome-specific probes are used. The green probe anneals to unique regions of chromosome 13, whereas the red probe is specific to chromosome 21. And on this slide, you'll see that there are three red signals, whereas there are only two green signals. So the three red signals are indicative of there being trisomy 21, or Down syndrome. Common microdeletion syndromes with consistent break points usually arise due to non-homologous recombination. And there are various commercially produced probes for these different microdeletion syndromes.

So here we have an image showing DiGeorge, or velocardiofacial syndrome, caused by a deletion at 22q11. The green probe is a satellite probe identifying chromosome 22. And the red probe is specific for the commonly deleted region. 

The normal image on the left shows two red signals for the 22q11 region. And the image on the right shows a single red signal for the 22q11 probe, indicating the presence of a deletion. And this one on the right can be seen in both the interphase and the metaphase nucleus. 

But one of the main uses for FISH is in the confirmation of array CGH finding. And this shows a 1q21 deletion, which has been picked up on array CGH. And that's shown on the left-hand side of this slide, where you have the ideogram, and then next to it a graph with a peak representing the 1q21 deletion. So to replicate the finding, a suitable FISH probe has been chosen. And the FISH image here on the right shows a single red signal representing the 1q21 region and therefore confirming the result. 

FISH can also be used in the identification of marker chromosomes. The karyotype here shows an extra piece of chromosome material, labelled here as mar, standing for marker. And the suspicion looking at the karyotype was that the marker came from chromosome 2. So FISH was used to confirm this.

The image on the left shows whole chromosome painting revealing that the marker chromosome is derived from chromosome 2, and that's painted red. And the right-hand image shows a green alpha satellite probe for chromosome 2, again confirming its origin.

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FISH is now widely used in hemato-oncology. And that's to aid diagnosis, estimate prognosis, and guide appropriate drug therapy. And the image shows the use of FISH in the diagnosis of chronic myeloid leukaemia, or CML. Probes to BCR and ABL regions are used. And this shows the classic translocation between chromosomes 9 and 22. And this is known as the Philadelphia chromosome, which is pathognomic of CML. 

The arrows point to the translocated chromosome, where the red and green signals can be seen to bind side by side. So having thought a little bit about how FISH works and then the clinical applications of FISH.

limitations of FISH

Let's think, If the “standard” FISH probe is between 100 and 200 kb, what do you think the limitations of FISH might be? One of the key limitations of FISH is that it might not detect deletions that are smaller in size than the FISH probe used. This is because the probe can “bridge” the deletion.
What other disadvantages, not related to size are there of using FISH?
As well as the limitations relating to size, there are also other disadvantages associated with using FISH:
 • The technique is time-consuming and relatively expensive.
• It requires a fresh blood sample (in a lithium heparin tube); the analysis can’t be undertaken on stored DNA.
• The technique is not useful to detect small tandem duplications where the signals will be overlapping and not therefore distinguishable from each other.
What information might FISH give you that is not available from other techniques to look for dosage abnormalities such as array CGH and MLPA? 
FISH will give you positional as well as dosage information. MLPA and array CGH will only tell you about the dosage of a given chromosome locus. 

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