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 identified2, 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 cancer4,5.
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 disease9,10.
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.
