Gone FISHing: new applications for an old technique

The use of fluorescent in situ hybridisation (FISH) has allowed for the advancement of molecular cytogenetics and kept research involving this technique at the cutting edge of science. However, FISH has a number of shortcomings, including a low resolution in its imaging, and restrictions in the sizes of probes that can be used. Extensions on this technique have therefore been developed in an attempt to improve upon the use of this process. An example of one such extension is the advent of comparative genomic hybridisation (CGH), a technique which utilises fluorescent probes to identify an increase or decrease in gene copy number that may be present within the genome in question. This development, along with a number of others, have potential to be powerful tools for several levels of investigation.

In a recent seminar given by Dr Miguel Pita, the use of fluorescent in situ hybridisation (FISH) was called into question. The primary focus of this talk was the application of fluorescent probes when using comparative genomic hybridisation (CGH). This is an important new technique within diagnostic medicine, with numerous other real world applications. CGH relies on the labelling of entire genomes to determine any amplification in gene copy number within an individual’s karyotype. This is a powerful tool, and has a number of future beneficial applications, which will be explored in more detail within this article.

Whilst completing my masters research project I was heavily reliant on the process of FISH to gain a greater understanding of the karyotype and any changes within the karyotype of Brassica napus (oilseed rape), as well as its parents Brassica oleracea and Brassica rapa. Whilst this was a crucial tool within this project, it was important for me to understand the limitations of this technique, which were highlighted within this seminar.

The birth of hybridisation techniques

The identification of tissues and the distribution of their chemical constituents was a key aspect of many early discoveries, and began by using various stains targeted (non-specifically) towards cellular structures. However, the lack in specificity of these stains limited their usefulness, with many moving away from these kind of techniques.

With no new major advances in the cytogenetic aspect of this field, molecular genetics became the main calling point for the majority of researchers. It took several decades for the advent of the fluorescent nucleic acid probes that we now commonly associate with molecular cytogenetics. However, probes of this kind were still incredibly primitive, relying heavily on radiolabelled probes, which came with the inherent drawbacks of using such chemicals. It therefore took a number of further advances before molecular cytogenetics was able to step into the limelight once again.

This began with the refinement of these in situ techniques, including a much greater increase in their sensitivity and resolution. Today, the majority of these cytogenetic approaches use fluorescently labelled sequences of DNA to detect areas with which they share homology, which is now commonly known as fluorescent in situ hybridisation (FISH)¹.

FISH is now used for a number of different purposes, including genetic counselling, within medicine and for general research purposes.

The procedure usually involved in the production of FISH probes

The process of FISH begins with the development of a probe. The sequence from which this probe is designed can be obtained in a number of ways, including PCR and micro-dissection of desired chromosomal sections. Once this sequence has been obtained, it is necessary to fluorescently label. These can be labelled either directly or indirectly, with the most common techniques used being nick translation or through the use of PCR utilising tagged nucleotides.

Once these sequences have been labelled, they are ready to be applied to chromosome preparations that have been suitably fixed upon a substrate. These spreads, and the effect that the labelled probes have upon them, can then be visualised and examined through the use of a fluorescent microscope¹.

The use of probes in this way has been an incredibly powerful tool within medicine and research, and has allowed for molecular cytogenetics to once again emerge as a leading investigative tool. The process of FISH is further explained in figure 1.

More examples of how FISH has been used

FISH is often used in medicine as a diagnostic tool. One such example of a disease that is used in this way is within downs syndrome. It is in this way that FISH probes are designed for sequences common only to chromosome 21. The detection of three fluorescent signals therefore confirms the presence of three copies of chromosome 21 (trisomy 21), therefore confirming a disease phenotype. This identification is not only limited to trisomy 21, with other trisomys also easily identified^{2, 3}.

FISH is also used to detect the presence of deletions that cause disease phenotypes such as prada-willi/angleman syndrome, as well as whole chromosome probes to detect the presence of translocations, including within certain forms of cancer^4,5.

However, whilst FISH is a very powerful tool, there is a still a limit on the resolution of this technique, with the size of the probe limited to above 100,000bp. There have therefore been a number of developments and expansions within this technique to extend the scope of its applications, either through increasing its resolution, or by applying this technique differently to which it was originally intended to extend its practical applications.

Expansions on the FISH technique

One such example where the scope of FISH has been extended by increasing the resolution that can be achieved from this kind of technique is illustrated by the use of tyramides. This works by adding proteins that are targeted for the required sequence (which can be much smaller than those required within standard FISH), followed by the addition of tyramides. These tyramides then hybridise around these small probes, thus amplifying the signals that can be produced. This process is illustrated further in figure 2.   Whilst this kind of hybridisation is much less accurate than the FISH technique, it does provide a more general idea of gene location, and at a much higher resolution. This kind of technique therefore has strong potential for preliminary diagnostics, as well as in paving the way for new methods of developing FISH with increasingly higher levels of resolution⁶.

Comparative genomic hybridisation (CGH)

Another example where the scope of FISH has been extended is with the development of comparative genomic hybridization (CGH). CGH is capable of revealing duplications and deletions within a patient genome when compared to a control genome. Within this technique, genomic DNA is extracted from the patient you wish to examine, followed be extraction of genomic DNA from a control patient. These two samples are then labelled with their own independently coloured label and mixed together. These two samples are then applied to a control metaphase spread (which is not derived from the patient that is being treated)⁷.

Visualisation in this way can see which coloured hybridisation is present in higher or lower quantities than others, as well as areas that are present in the same quantities (visualised by a mix of the two colours). This indicates any change in gene copy number variation within a patients, including whole chromosome arm translocations, smaller translocations, and duplications in much smaller numbers.

Whilst much recent research has been geared towards the use of bioinformatics in order to determine genetic abnormalities, this would be an unrealistic approach towards diagnosis in humans. This is mainly due to the fact that diagnosing in this way would require the sequencing of whole individual organisms, which would be time consuming and expensive. The use of CGH is a much easier and more powerful tool for initial visual identification of a disease phenotype, therefore reducing diagnosis times within patients and allow for much faster treatment; greatly benefitting suffers of a number of diagnosable treatments.

CGH is also emerging as a powerful tool for the genetic screening of embryos to detect abnormalities in their ploidy level. Changes in chromosome number lead to a large number of miscarriages, as well as conditions such as trisomy 21, causing downs syndrome, as mentioned previously. Prior to the advent of CGH, detection of chromosomal abnormalities was a much longer and more arduous process, therefore highlighting one particular area where the application of CGH has eased investigation and diagnosis⁸.

CGH also has applications beyond those involved purely in healthcare. Two examples that were explicitly mentioned within this seminar are as follows.

The first example involved comparing the karyotypes between the prave vole, which is monogamous in nature, and the meadow vole, which is non-monogamous in nature. In this case, CGH was used to determine whether chromosomes may have recently fused to try and provide some explanation behind this difference in behaviour. Being able to further understand this kind of genetic control could have important implications regarding the understanding of these animals as a species.

The second example given was also a comparison between two different animal species. In this case, C. parallelus erythroptic and C. parallelus. These two species exist in two distinct geographical areas, with the existence of a hybrid zone between the two species. The resultant progeny produced between these two species within the hybrid zone are infertile. It is thought this infertility is due to an increase in the number of bacteria within that can survive and thrive within these hybrids. CGH has therefore been employed in an attempt to determine any chromosomal insertions that may occur between these hybrids to determine why it is that bacteria can thrive better within this hybrid environment. This is not only important to further understand this species, but could also give us a greater understanding over the types of genes that are important to fight off bacterial infections, which could have human implications with further research.

The use of CGH is therefore an important step in advancing the field of cytogenetics. However, it is also crucially important that other techniques of this kind are also developed and enhanced upon in order to ensure that quick and easy diagnostic medicine can maintain its momentum, reduce the time where a patient is unsure of their diagnosis, and meet the needs of all those involved in the administering of treatment. The specific use of CGH and other more classical techniques in tandem also have optimistic applications in understanding oncogenic phenotypes, taking us one step closer to personalised treatment in response to this disease^9,10.

If techniques like this one, and others mentioned within this article, continue to develop and be expanded upon, cytogenetics can remain an important tool for a wide range of diagnostic purposes. It is important that cytogenetics is not left behind by the advent of more molecular based tools, as it is clear that cytogenetics has not given all that it is able to give.

What are your thoughts on this seminar topic? Have you learnt about a new technique that maybe you hadn't heard of before? Comment below with any questions and don't forget to +1 if you enjoyed this article. Thanks for reading!

References:

1.      Levsky, M.J. Singer, R.H. (2003) Fluorescence in situ hybridisation: past present and future. J Cell Sci. 116. P2833-2838.  

2.      Bishop, R. (2010) Applications of fluorescence in sity hybridisation (FISH) in detecting genetic aberrations of medical significance. Bioscience Horizons. 3 (1): p85-95.

3.      Pinkel, D. Landegent, J. Collins, C. Fuscoe, J. Segraves, E. Luxas, J. Gray, J. (1988) Fluorescence in situ hybridisation with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. PNAS. 85 (23) p9138-9142,

4.      Fox, J.L. Hsu, P.H. Legator, M.S, Morrison, L.E, Seelig, S.A. (1995) Fluorescence in situ hybridisation: powerful molecular tool for cancer prognosis. Clin Chem. 41 (11): p1544-9.

5.      Kearney, L. Shipley, J. (2012) Fluorescence in situ hybridisation for cancer-related studies. Methods Mol Biol. 878: 149-74.

6.      Van Gijlswijk, R.P. Wiegant, J. Raap, A.K. Tanke, H.J. (1996) Improved localization of fluorescent tyramids for fluorescent in situ hybridisation using dextran sulphate and polyvinyl alcohol. J Histochem Cytochem. 44 (4) p389-92.

7.      Pinkel, D. Albertson, D.G. (2005) Comparative genomic hybridisation. Genomics and Human Genetics. 6. P331-354.

8.      Frenny, S. Harsh, S. Kiumarri, P. Stuti, T. Manisha, D. Patel, B. Jayesh, S. (2014) 1 (1) p 3-9.

9.      Cremer, T. Cremer, C. Lichter, P. (2014) Recollections of a scientific journey published in human genetics: from chromosome territories to interphase cytogenetics and comparative genome hybridisation. Hum Genet. 133: 403-416.

10.  Lall, M. Saviour, P. Puri, R. Paliwal, P. Mahajan, S. Verma, I. (2014) Molecular cytogenetics. 7: p9-13.