Fluorescence in situ hybridization (FISH) uses fluorescent probes to label specific regions of nucleic acid sequence in fixed cells or tissue sections. It is used in cancer genomics, in aging and developmental biology, and in studies of mechanisms of gene transcription, among a variety of other disciplines in which genomic structure and morphology are of import. FISH can synergize with other cytological techniques ranging from immunohistochemistry to electron microscopy.
While next-generation sequencing (NGS) may have cut in to the role of FISH— NGS has allowed for the identification of more and more, and smaller and smaller, insertions and deletions (indels) — “you really don’t know what the heterogeneity of those variants is, and whether there is a correlation of a histological feature of the tissues,” says Cristina Montagna, scientific director of the Albert Einstein College of Medicine Molecular Cytogenetic Core. “You still need to go back to FISH to better understand what you have discovered with your NGS tool.”
The protocols, the labeling, and the types of probes used in routine FISH today are largely the same as they were when the technique was developed in the early 1980s; what has improved is the imaging, thanks largely to more sensitive (digital) cameras and brighter dyes, Montagna adds. In this article, we will delve into how that routine is accomplished, as well as a few techniques that push those boundaries.
BAC to the Basics
FISH can be generic, as when using DNA dye like 4',6-diamidino-2-phenylindole (DAPI) to fluorescently label all the chromosomes (a technique often used as a counter-stain). But the term more often applies to the broad (looking at large chromosomal regions, such as the telomere or centromere, up to whole chromosome) or locus-specific recognition of chromosomal DNA with fluorescently labeled nucleic acid probes. In any case, it takes a lot fluorophores, on many probes, covering a considerable stretch of DNA, to generate enough signal to be detected by a fluorescence microscope.
By far the most common way to generate such probes is to start with bacterial artificial chromosome (BAC), fosmid, cosmid, or yeast artificial chromosome (YACs) clones complementary to the region of interest, which are then nick-translated. The DNA is first nicked with a nickase; the exonuclease activity of a DNA polymerase then removes nucleotides from the DNA, and the polymerase activity replaces them with a combination of untagged and tagged deoxynucleotides (dNTPs). Adjusting the nickase concentration allows the user to optimize the product size, while adjusting the ratio of the tagged to untagged dNTPs allows for more or fewer fluorophores to be incorporated into the probes.
Two to three BACs will fully cover an average gene, says Christine Shaw, senior research technician at the Memorial Sloan Kettering Cancer Center (MSKCC) Molecular Cytogenetics core facility.
Many facilities like MSKCC “still make all our own buffers and reagents from scratch, and our methods are very classical,” she says.
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Others prefer to purchase nick translation kits from one of a variety of vendors. These may differ in whether tagged dNTPs are included in the kit or sold separately. The tagged dNTPs may also differ—some, for example, may have the fluorophore directly attached, while others offer amine-modified dNTPs to which a dye can be attached following their incorporation into the DNA. Most kits’ protocols recommend overnight labeling; Enzo Life Sciences “has streamlined the protocol, so the reaction is finished in an hour,” says Jack Coleman, the company’s director of biochemistry.
BAC probes tend to have repetitive sequences, which can cause nonspecific hybridization in a FISH assay. Reagents such as Cot DNA can be used to compete out these interactions, thereby reducing the background signal.
Oligo Probes
In contrast, by using bioinformatically chosen oligonucleotide probes, repetitive sequences can be all but eliminated from the probe design. “Oligo probes involve, basically, replicating in vitro, usually through PCR amplification, shorter, sequence-specific segments,” explains Katherine Geiersbach, senior associate consultant in the department of laboratory medicine and pathology at Mayo Clinic.
Between 50 and 100 such probes are typically needed to build up sufficient signal to visualize a target. While oligo probes for many common targets may be available off-the-shelf, it takes a fair amount of time and expertise, not to mention cost, to design and produce custom probes. Thus oligos are still not a mainstay of routine use in the research and discovery setting.
Yet when they are called for, several options are available beyond homebrew, including those with a standard DNA backbone, or a backbone composed of modified nucleotides such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), bridged nucleic acids (BNAs), or morpholinos. Each of these may offer advantages in terms of, for example, stability, binding conditions, and kinetics.
Montagna uses custom-synthesized LNAs from Exiqon “because I found the information through a variety of papers I was reading. I did not explore other possibilities—in my opinion, their probes are relatively affordable and are good, and I did not see the need to look any further.”
Paint by Numbers
Chromosome painting is a technique in which probes allow for visualization of a chromosome in its entirety. To analyze copy number of a particular chromosome—to look for a trisomy, for example—then just paint for that chromosome. “Why not just use an enumeration probe (which stains the centromere)?,” Asha Multani, co-director of the University of Texas’ MD Anderson Molecular Cytogenetics Facility, asks rhetorically. “Sometimes the chromosome could be broken and a fragment is there, but you don’t have the centromere—in that case it would be more informative to use the paint probe. The technique can also be multiplexed by using differently-labeled probes for different chromosomes.”
Another option for multiplexing is Applied Spectral Imaging’s spectral karyotyping (SKY): all the chromosomes can be simultaneously painted with different spectral dyes, which are then distinguished by means of an interferometer and software, Multani explains. “When you do G banding, you can’t tell subtle translocations—you don’t know where the piece came from. Especially in tumors, chromosomes are so rearranged you don’t know which has gone and fused with the other chromosome. With SKY you can actually tell those translocations.”
On a more experimental front, “the latest, hottest thing is to be able to look at thousands of different [RNA] sequences in a single cell” with FISH, says Robert Singer, professor of cell biology at the Albert Einstein College of Medicine, referring to work from the labs of California Institute of Technology’s (Caltech’s) Long Cai, Harvard University’s Xiaowei Xhuang, and others. “At the present time it’s technically demanding, but not out of the range of a smart grad student.”
The latest, hottest thing is to be able to look at thousands of different [RNA] sequences in a single cell with FISH.
And among Singer’s own recent contributions is the use of fluorescently labeled, nuclease-deficient CRISPR/Cas9 complexes as probes to target genetic sequences in situ without global denaturation, allowing researchers to better preserve the structural features of the DNA. “In CASFISH [Cas9-mediated fluorescence in situ hybridization] there is no need to melt the DNA because the Cas9 guide complex recognizes the double-stranded form, so it’s a much gentler procedure,” he explains. “The fact that you can label a protein makes it much cheaper … and you can put many, and brighter, fluors on the protein— there’s much better chemistry with those probes.”
Even as its edges bleed, FISH’s core technology has remained fairly stable. It continues to be a gold standard, with journal reviewers asking to see copy number by FISH, for example, says Multani. After all, an image is worth 1000 words, and who knows how many NGS data points.
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