FISH: the importance of in situ probe design

Fluorescence in situ hybridisation (FISH) is an invaluable tool for the diagnostic and prognostic analysis of patient samples, providing a routine means of identifying genome duplications, deletions and rearrangements associated with disease.

The central component of any FISH assay is the probes. These are pools of fluorescently labelled DNA or RNA sequences designed to hybridise to target sequences, and that can subsequently be identified through imaging techniques such as fluorescent microscopy.

Discover how FISH can be used in cancer diagnostics and research

In situ probe design can be a challenging task for many labs, requiring significant time, money and expertise. However, without well-designed probes, your FISH experiments could be futile. But it’s not all doom and gloom, check out our five tips on what to look for when it comes the design of your in situ probes.

1. Check for commercially available probes

Before you spend time, effort, and expense designing and optimising probes for your FISH assay, it’s worth checking whether any suitable probes have already been designed and/or are commercially available. That way, you save precious time and money by minimising the validation process.

Suppliers, such as OGT, offer an extensive range of high quality, reliable and easy-to-use DNA FISH probes for many clinically-important applications. OGT’s CytoCell^® probe range includes probes for the accurate detection of many haematological malignancies, common prenatal chromosomal disorders, and some of the rarest human genetic syndromes as well as the assessment of genetic aberrations in solid tumour samples.

Eight of OGT’s CytoCell FISH probes, designed to detect prenatal trisomy 13 & 21 and acquired cancer-related chromosome alterations, were recently granted IVDR certification, becoming the first FISH probes to achieve this. The IVDR certification means that customers can be confident in the continued quality, safety and performance of OGTs products and manufacturing processes.

Discover OGT’s range of CytoCell FISH probes and view our eight IVDR FISH probes

2. Choose BAC clone probe design for unparalleled coverage

There are two main classes of probe that can be used for FISH: Probes based upon bacterial artificial chromosome (BAC) clones or oligo probes. BAC clones are distinct fragments of human chromosome that were originally cloned for sequencing as part of the Human Genome Project, but have since become highly valuable for FISH applications.

BAC clones can be substantial in size, averaging several hundred kilobases¹, plus the entire BAC can be labelled with a fluorophore making for a relatively simple probe design workflow. In contrast, oligo probes are short, synthetic DNA fragments that are designed to specifically target a pre-determined region of interest.

Due to their significantly greater size, BAC probes offer greater coverage versus oligo probes. As such, BAC clones are able to target multiple genes or entire gene clusters, making them the ideal choice for detecting large genomic rearrangements, chromosomal aberrations and copy number changes. In light of the unparalleled coverage provided by BAC clone probes, all of OGT’s CytoCell FISH probes are based upon BAC clones. 

3. Make use of published data

Scientific papers are a valuable resource for in situ probe design, especially when searching for genetic aberration(s) associated with your disease of interest. By searching literature databases such as PubMed, you can find a wealth of study data highlighting clinically relevant genetic defects. From this, you can begin to compile a list of candidate aberrations to target with your FISH probes.

Once target aberrations have been identified, publicly available genomic databases such as Ensembl or UCSC can be leveraged to pinpoint BAC clones that could be included in your probe design. Examples of useful BAC clones could be those located at either side of a translocation breakpoint or a BAC clone inside a region that is known to be deleted or amplified.

Once you have carefully selected the appropriate BAC clones for your FISH assay, these can be purchased from BAC clone libraries or commercial vendors. It’s highly recommended to then validate the specificity of any BAC clones purchased by confirming their identity and integrity via sequencing and pilot FISH assays with control samples.

4. Select the right fluorophore for your application

The myriad of fluorescent labels available for probe labelling can leave many of us feeling uncertain. There are many considerations to be made when selecting a fluorophore for your FISH probe, so here are some key factors to help you feel confident in your decision:

• Analysis instrument: Your specific instrument will have features that affect your choice of fluorescent label such as excitation source, optical filters, and sensitivity. Check the spectral properties of your prospective fluorophore matches your machine’s capabilities.
• Absorption-emission spectra: While the absorbance of a dye refers to how much visible or UV light it absorbs, the emission value shows at which wavelength it emits light radiation out. Since each fluorescent dye has its own absorption and emission value, it is important to ensure compatibility with your instrument and assay.
• Stokes shift: This is the difference in energy between the excitation and emission maximum. Generally, a larger stokes shift is favourable because it is easier to distinguish between the excitation and emission wavelengths. It is particularly important in multiplex applications because one emission wavelength of one fluorophore may overlap, and therefore excite, another fluorophore in the same sample.
• pH-sensitivity: Fluorophores show varying sensitivity to alkaline or acid pH conditions. Ensuring your fluorophore will not degrade in your test environment is critical to a successful FISH experiment.

5. Consider using an expert third party

Depending on your application, how well studied your disease of interest is, and the level of expertise in your lab, FISH probe design and subsequent optimisation steps can take substantial time and effort. Expert third parties offer custom FISH probe design and manufacture services. For example, through its CytoCell myProbes^® service, OGT experts can work with you to deliver optimised probes that meet your unique requirements – whether you need a simple modification of an existing catalogue product or require an entirely new and unique probe set.

Find out more about OGT’s CytoCell myProbes service

Gone FISHin’

Since its inception, FISH technology has diversified. There are now countless different diagnostic tests in which scientists can exploit FISH and many different diseases and disorders can be studied. Therefore, while the tips and tricks above serve as a great platform for initial in situ probe design, each application will have its own nuances. Design guidelines will vary based on the particular assay you are using and the type of variation you’re targeting – but following these guidelines will provide a good start.

If you still need help designing your FISH probes, at OGT we’re here to help with our CytoCell myProbes^® custom FISH probe design and manufacture service. Alternatively, why not see if your needs could be instantly met with our wide range of CytoCell FISH probes optimised for haematological malignancies and the assessment of genetic aberrations in solid tumour samples.

References

1. Liu C, Guo Y, Lu T et al. Construction and preliminary characterization analysis of Wuzhishan miniature pig bacterial artificial chromosome library with approximately 8-fold genome equivalent coverage. Biomed Res Int. (2013) 587493.