Fluorescence in situ hybridization technique

Fluorescence in situ hybridization technique

The introduction of FISH (fluorescence in situ hybridization) marked the beginning of a new era for the study of chromosome structure and function. As a combined molecular and cytological approach, the major advantage of this visually appealing technique resides in its unique ability to provide an intermediate degree of resolution between DNA analysis and chromosomal investigations while retaining information at the single-cell level. Used to support large-scale mapping and sequencing efforts related to the human genome project, FISH accuracy and versatility were subsequently capitalized on in biological and medical research, providing a wealth of diverse applications and FISH-based diagnostic assays. The diversification of the original FISH protocol into the impressive number of procedures available these days has been promoted throughout the years by a number of interconnected factors: the improvement in sensitivity, specificity and resolution, together with the advances in the fields of fluorescence microscopy and digital imaging, and the growing availability of genomic and bioinformatic resources. By assembling in a glossary format many of the “acronymed” FISH applications published so far, this review intends to celebrate the ability of FISH to re-invent itself and thus remain at the forefront of biomedical research.
The introduction of fluorescence in situ hybridization (FISH) almost 30 years ago marked the beginning of a new era for the study of chromosome structure and function. Conceptually, FISH is a very straightforward technique that essentially consists in hybridizing a DNA probe to its complementary sequence on chromosomal preparations previously fixed on slides. Probes are labeled either directly, by incorporation of fluorescent nucleotides, or indirectly, by incorporation of reporter molecules that are subsequently detected by fluorescent antibodies or other affinity molecules. Probes and targets are finally visualized in situ by microscopy analysis. As a combined molecular and cytological approach, the major advantage of this visually appealing technique resides in its unique ability to provide an intermediate degree of resolution between 

DNA analysis

 and chromosomal investigations, while also retaining information at the single-cell level. FISH gained widespread recognition as a physical mapping technique to support large-scale mapping and sequencing efforts related to the human genome project; however, its accuracy and adaptability were simultaneously, or soon after, exploited in other areas of biological and medical research. As a result, a wealth of diverse applications and FISH-based diagnostic assays have been developed within different fields of investigation, including clinical genetics, neuroscience, reproductive medicine, toxicology, microbial ecology, evolutionary biology, comparative genomics, cellular genomics, and chromosome biology. The diversification of the original FISH protocol into the impressive number of procedures available these days has been promoted through the years by a number of interconnected factors, such as the improvement in sensitivity, specificity, and resolution of the technique, brought about by a better understanding of the chemical and physical properties of nucleic acids and chromatin, together with the advances in the fields of fluorescence microscopy and digital imaging, and the growing availability of genomic and bioinformatic resources. Here, we have assembled in a glossary format many of the FISH applications published so far. Although we intend this review to celebrate the versatility of this technique, it is of course impossible to cover every modification of FISH, and therefore we have limited ourselves to variants that are named by combining a prefix with the acronym FISH. As seen in the many flavors of FISH described in the following, this flexibility has allowed the underlying technique to remain at the forefront of biomedical research over the last three decades.
ACM-FISH is a multicolor FISH assay for the simultaneous detection of numerical and structural chromosomal abnormalities in sperm cells (1). The abbreviation ACM refers to the concurrent hybridization of 

DNA probes

 for the alpha (centromere), classical (1q12), and midi (1p36.3) satellites of chromosome 1 for the specific detection of duplications and deletions of 1pter and 1cen, and for the identification of chromosomal breaks within the 1cen-1q12 region. The ACM technique originated from the integration of technical aspects and biological findings that emerged from previous FISH investigations of chromosome rearrangements in sperm (2), as well in other cell types (e.g., the assessment of breaks in 1q12 in human lymphocytes)(3,4). Its application has led to significant discoveries in the occurrence of chromosomal abnormalities in the sperm of healthy men, showing that spontaneous structural defects are more frequent than numerical ones and that chromosomal breaks preexist in human sperm before fertilization, and also providing evidence for reproducible donor differences in breakpoint locations within 1q12 (1). The assay has also been successfully used for the analysis of sperm of infertile men to show that oligozoospermic men carry a higher burden of transmissible chromosome damage; these results raising the question of possible elevated levels of chromosomal defects following intracytoplasmic sperm injections (ICSI) treatments (5).
Arm FISH is a 42-color M-FISH variant (see below for M-FISH) that allows the detection of chromosomal abnormalities at the resolution of chromosome arms (p- and q-arms of all 24 human chromosomes, except the p-arm of the Y and acrocentric chromosomes) (6). The protocol combines a commercially available M-FISH kit with an arm kit or a set of chromosome arm-specific painting probes (7). It is a straightforward, but significant, improvement to the standard M-FISH technique, the most obvious advantage being the increased resolution to the level of chromosome arms and the resulting ability to detect pericentric inversions. The assay has been successfully applied to reveal widespread chromosomal instability in glioma cell lines (8).
CARD-FISH, which stands for catalyzed reporter deposition-FISH, refers to the signal amplification obtained by peroxidase activity through the deposition of a large number of fluorescently labeled tyramine molecules in which the horseradish peroxidase (HRP)-labeled probe has bound (see also T-FISH). Improvements have been made to the technique to aid the delineation of bacterial sequences, with one aspect of improvement being collecting bacteria on filters (9–11). There has also been a combination of CARD-FISH with microautoradiography that has been termed MICRO-CARD-FISH (12–14). Tritium is the radioisotope that is usually used and is taken up by active cells as 3H-aspartic acid. The filters on which the bacteria are captured are submitted to CARD-FISH, and they are then placed onto photographic emulsions. Cells can be assessed using a fluorescence microscope that has transmission light in addition. This allows both fluorescent, positive cells to be viewed by excitation of UV light and co-localization of silver grains by white light.
Cellular compartment analysis of temporal (cat) activity by FISH is an ingenious experimental approach devised to investigate the dynamic interactions of neuronal populations associated with different behaviors or cognitive challenges (15). The method, based on RNA-FISH on cryosections followed by confocal analysis, was originally applied to study the environment-specific expression of the neural activity-regulated, immediateearly gene (IEG) Arc, and to monitor its cellular and subcellular distribution in the whole brain in rat (16). As a functional brain imaging technique, the uniqueness of catFISH resides in its ability to confer both temporal and cellular resolution to the analysis of gene expression patterns in brain, an important combination for the study of the dynamics of information processing.
CB-FISH involves hybridization on binucleated cells in which cytokinesis has been blocked by treatment with cytochalasin-B (CB). The term CB-FISH was coined by a research group investigating the mechanism by which the ratio of mosaic diploid cells in vivo increased in trisomy 21 cases (17). However, protocols combining FISH with the CB blocking assay for the cytological analysis of micronucleation and aneuploidy events had already appeared in a number of earlier molecular cytogenetic studies (18–21), including an investigation of chromosome 21 malsegregation in Alzheimer’s patients (22). Analysis of the chromosomal content of micronuclei can be facilitated by combining the standard CB-FISH protocol with the 24-color SKY technology, since in one hybridization step, the DNA from any chromosome within the micronuclei can be identified (23,24) (see below for M-FISH and SKY).
Chromosome orientation or CO-FISH is the name given to a FISH technique that uses single-stranded DNA probes to produce strand-specific hybridization. The technique relies on labeling by 5'-bromodeoxyuridine (BrdU) incorporation one strand of the sample cells’ DNA during S-phase. Metaphase chromosomes are prepared a number of hours after the BrdU pulse. Following Hoechst staining and UV irradiation, the newly synthesized DNA becomes nicked at the sites of BrdU incorporation. These nicks are enlarged using ExoIII, and the newly replicated DNA is removed, leaving the parental strand as a single-stranded template for the hybridization procedure. Initially, CO-FISH was designed to determine the orientation of tandem repeats within centromeric regions of chromosomes (25,26). This technique had also been useful in assessing aspects of translocated chromosomes, specifically Robertsonian (27,28), and also chromosomal inversions, since an inversion obviously changes the orientation of the involved chromosome segment (29,30). For a review on CO-FISH, see Bailey et al. (31).
The prefix COBRA stands for combined binary ratio. This particular FISH protocol brings together combinatorial labeling with ratio labeling (32). The ratio labeling method allows different ratios of label to distinguish between probes. This permits the use of fewer fluorochromes to produce more pseudocolors, allowing the resolution of more than 24 colors within a specimen. The novel aspect of the COBRA technique is that two sets of probes are ratio-labeled identically with three fluorochromes (usually two sets of 12 chromosomes for a human karyotype), but then one set is further labeled with an additional fourth fluorochrome. Indeed, a further fifth fluorochrome can be used to make up to 48 color combinations for differential painting of human chromosome arms (33). The capacity to view more than 24 colors allows the delineation of additional sequences such as viral genome inserts or single-copy genes (34). For a comprehensive review of the technique see Raap and Tanke (35).
Although COD-FISH is an abbreviation that has been used to describe three different hybridization techniques, the most common use is for chromosome orientation and direction-FISH. This protocol is similar to CO-FISH (see above), but when combined with the information about the directional organization of telomeric sequences, the technique can be termed COD-FISH (30). COD-FISH can also stand for concomitant oncoprotein detection-FISH, a technique to visualize not only the loci signals for a particular oncogene, but also the protein product derived from this gene. The development of such a protocol was aimed at performing quantification of gene copy number and amount of protein product to help elucidate interesting mechanisms involved in the transcription and translation of a particular message (36). Others have called this type of regimen Immuno-FISH (see below), but the COD protocol does add a semiquantitative aspect to it. Tubbs and colleagues went on to improve their method using nanogold visualized with bright field microscopy (37). Another technique that has been termed COD-FISH is the combined CaCO3 optical detection-FISH, in which FISH is used to detect calcifying microrganisms in open ocean (38).
COMBO-FISH is a method with no requirement for sample denaturation prior to hybridization. The prefix COMBO stands for combinatorial oligonucleotide. The technique utilizes sequence information to identify regions of the genome where there are stretches of purines or pyrimidines and uses homopurine or homopyrimidine probes that are able to form triple helices with duplex genomic DNA (39). Homopurine or homopyrimidine regions of DNA 14 bp in length or more compose 1% to 2% of the human genome, with an average of 200 of such stretches in a 250-kb segment of the genome. Accordingly, specific probe sets can be constructed to target genomic regions of interest in that size range. The omission of the denaturation step makes the hybridization procedure less harsh on nuclear architecture, rendering this technique ideal for three-dimensional (3-D) analysis of genome organization (40,41).
Comet-FISH is a combination of the comet assay and FISH analysis. The comet assay, also called single-cell gel electrophoresis or the single-cell gel test, is used to evaluate the amount of DNA breakage within single cells by running the DNA out of the nuclei into an agarose gel. The combined Comet-FISH method, consisting in releasing, by electrophoresis, the DNA onto agarose-coated microscope slides prior to in situ hybridization, allows specific sequences to be delineated in the comet head or tail, thus permitting the assessment of whether specific genomic regions are sensitive to DNA damage and breakage (42,43). With this technique, researchers have demonstrated that DNA damage susceptibility is associated with the gene density of a chromosome rather than the chromosome size (43). Further, damage to specific genes can be detected (44–47). The sensitivity of telomeres to damage has also been assessed successfully using this method (48). See Rapp et al. (49) for an overview of the technique.
Cryo-FISH is a promising in situ hybridization technique that makes use of ultrathin cryosections (150 nm thick) of well-fixed, sucrose-embedded cells. The technique was recently devised and successfully applied to the study of spatial interrelationships of chromosome territories in the cell nucleus (50), a muchdebated aspect of chromosome organization in interphase. By combining a robust cell fixation procedure and ultracryomicrotome sectioning with two-dimensional (2-D) microscopy analysis (wide-field), this innovative technique maintains to preserve chromatin nanostructure while simultaneously presenting with a better efficiency of hybridization and resolution than canonical 3-D FISH, and provides an alternative, and possibly more userfriendly approach, to the study of genome organization in the nuclear context. Cryo-FISH was also recently used to validate results obtained by chromosome conformation capture on chip (4C) technology to demonstrate long-range chromosomal interactions of functional significance (51).
D-FISH is an enhanced version of the fusion signal-FISH protocol for the detection of recurring chromosomal translocations in hematological malignancies (see below for fusion-signal FISH). The prefix D stands for double fusion, since in this particular protocol the use of two (or two sets of) differentially labeled, large probes, each spanning one of the two translocation breakpoints, allows the simultaneous visualization of both fusion products, significantly reducing the impact of false-negative results, a reason of concern in single fusion FISH. D-FISH was initially devised to improve detection of the double BCR/ABL fusioBCR/fusion in chronic myeloid leukemia (CML) patients (52,53). Subsequently, D-FISH probes were developed for the 8;21 translocation and used for the assessment of minimal residual disease in acute myeloid leukemia (AML) patients during remission (54) and also for the visualization of PML/RARA double fusion in acute promyelocytic leukemia (APL) (55,56), the DEK/CAN double fusion resulting from t(6;9) in AML (57), and the PBX1/E2A double fusion in pediatric patients with acute lymphoblastic leukemia (ALL) (58). A wide range of probes for D-FISH, as well as Fusion- Signal and Split-Signal FISH for the chromosomal analysis of hematologicalcancer, are now commercially available.
DBD-FISH stands for DNA breakage detection FISH, a technique developed by Gosálvez and colleagues (59,60). Basically, this adaptation of the FISH procedure permits any sites of DNA damage/breakage in the sample genome to be analyzed in situ by means of an alkali DNA unwinding solution and protein removal. Cells are normally stabilized in agarose beads, but the technique can be applied to DNA comets (61). The incubation with the unwinding buffer leads to the presence of single-stranded DNA in the sample that can be hybridized with the appropriate probes (62,63). The technique has been used successfully to test the DNA fragmentation levels in sperm samples (64).
E-FISH is a BLAST-based FISH simulation program able to accurately predict the outcome of hybridization experiments. The program was developed as one of the bioinformatics resources to be available from the Database of Genomic Variants, aimed at simplifying the choice of appropriate genomic probes for hybridization experiments and facilitating the interpretation of the results (65). This virtual FISH approach includes a repeat masking step mimicking in silico the COT-1 blocking of repetitive sequences. The program is freely accessible at projects.tcag.ca/efish.
Fiber-FISH is a technique that allows high resolution mapping of genes and chromosomal regions on fibers of chromatin or DNA, permitting physical ordering of DNA probes down to a resolution of 1000 bp, and that also allows assessment of gaps and overlaps in contigs and analysis of segmental duplications and copy number variants. In practice, the method consists of releasing chromatin/DNA fibers from cell nuclei, usually by means of salt or solvent extraction, and stretching and fixing them on a microscope slide prior to hybridization. Similar variants of the technique, in which the chromatin is run down the slide and is stretched by fluid flow, were initially set up more or less concomitantly by different research groups (66–68). However, the specific Fiber-FISH terminology was introduced slightly later (69,70). A significant improvement in terms of DNA stretching uniformity and reproducibility was provided by the implementation of the molecular combing protocol (71), which uses the action of a receding air/water meniscus to extend and align DNA molecules attached at one end to a glass surface.
In the Flow-FISH technique, as described by Lansdorp and colleagues in 1998 (72), PNA-labeled telomere probes are used to visualize and measure the length of telomere repeats, as in the Quantitative-FISH technique (see below for Q-FISH and PNA-FISH), but the analysis combines in situ hybridization with flow cytometry for measurement of the telomeric signals from cells in suspension. This permits large numbers of cells to be analyzed rapidly. Lansdorp and his collaborators have developed this technique, and his and other laboratories have used it for a number of different cell types and clinical applications (73). Indeed, Flow-FISH has been used in aging studies (74–76), telomere maintenance (77,78), and in clinical applications for ex vivo suspension cells (hematopoietic) (79–84). Also, combining telomere Flow-FISH with fluorescent immunodetection of cell surface markers has advanced the understanding of the behavior of stem cell populations (80,85–88). Researchers have expanded the Flow-FISH technique to permitassessment of different strains of bacteria (89,90).

FLow-FISH technique using 3rd generation nucleic acid (BNA) has high binding specificity and nuclease resistance. It can be use to replace PNA probe.
Fusion-Signal FISH
The Fusion-Signal FISH technique was initially devised for the identification of the 9;22 Philadelphia translocation in peripheral blood and bone marrow cells of CML patients to detect minimal residual disease after bone marrow transplantation (91). BCR and ABL gene fragments, each flanking one of the two breakpoints, were used as probes for the detection of the BCR/ABL fusion product, hence the fusion-signal appellation. Since then, sets of probes to detect fused gene signals originating from a range of critical translocation events in hematological malignancies, for instance the PML and RARA fusion product resulting from t(15:17) in APL (92), have been designed by different research groups. With FISH assays available for CML and APL, as well as AML (93,94), non-Hodgkin’s lymphoma (95,96), mantle cell lymphoma (97), childhood B-lineage acute lymphoblastic leukemia (98–100), and infant leukemia (101), it would be difficult to deny the major impact of the fusion signal technique as a diagnostic and prognostic tool for blood cancer. However, soon after its conception, concerns started to emerge that the interpretation of the results was complicated by the variable occurrence of false-positive and false-negative signals (102–104). To overcome these difficulties, an ingenious dual-fusion variant of the technique was devised for the detection of the 9;22 translocation in CML (52). The protocol involves the use of large probes, spanning the two breakpoints, for the simultaneous visualization of both fusion signals BCR/ABL and ABL/BCR. This modified and improved version of the fusion signal technique was named D-FISH (see above).
Halo-FISH is an acronym that describes FISH performed on cells that are first permeabilized and then extracted with high salt to remove soluble proteins (105,106). Indeed, chromatin/DNA that is not fixed to an internal structure within cell nuclei is released, forming a halo around a residual nucleus. FISH can then be performed on these preparations using any type of probe to delineate specific DNA sequences. Researchers have used a-satellite (106), telomeres (107), scaffold attachment regions (SARs) (108), matrix attachment regions (MARs) (109), gene loci (105,110,111), and whole chromosomes (112,113). DNA halo preparations can be used for high-resolution mapping, since such long extended loops of DNA are created (114). A number of groups have used DNA halo preparations to analyze sperm chromatin, as it makes it easier to access sperm DNA, which is normally very compact due to its association with protoamines. Some have even described this type of analysis as SpermHalo-FISH (115), whereby sperm nuclei are spun onto glass microscope slides and treated with dithiothreitol for permeabilization followed by high salt.
Harlequin-FISH is a method for cell cycle-controlled chromosome analysis in human lymphocytes that allows a precise quantification of induced chromosome damage for human biodosimetry purposes (116,117). The approach combines FISH painting with differential replication staining after BrdU treatment of lymphocyte cultures (or harlequin staining). The principle, on which differential replication staining is based, is that after two rounds of replication in the presence of the base analog BrdU, sister chromatids will stain differentially (with either Giemsa and/or fluorescent dyes) (118,119). This allows the identification of the two chromatids and the observation of sister chromatid exchanges (SCEs), that after a few cell divisions confer to the chromosomes an asymmetrically striped appearance, to which the term harlequin refers. Since for accurate cytogenetic measurements of genetic damage, cells must be analyzed in their first mitosis following exposure, the most relevant aspect of the harlequin technique in combination with FISH for biodosimetry studies is that, according to sister chromatid staining patterns, cells in different division cycles can be distinguished, allowing chromosomal analysis to be carried out selectively.
Immuno-FISH is a combination of two techniques, one being standard FISH, either on flattened chromosome preparations (2-D FISH) or on three-dimensionally preserved nuclei (3-D FISH), and the other indirect or direct immunofluorescence. The latter technique permits the visualization of antigens within the sample, so that both DNA and proteins can be analyzed on the same sample. It is often used to investigate co-localization of genomic regions with proteinaceous entities within interphase nuclei such as nucleoli (120) or promyelocytic leukemia (PML) bodies (121). Not all antigens will be preserved after the various steps in the FISH protocols, but it is possible to apply the primary or primary and secondary antibodies and proceed with a fixation step prior to the FISH procedure (122). The term Immuno-FISH was first coined by Brown et al. in 1997 (123), in which co-localization of active or inactive gene loci were assessed with nuclear structures containing the Ikaros protein, although others had combined the two methodologies previously for RNA-FISH with splicing speckles (124) and for anti-CENP C staining in combination with a-satellite probes (125). The combination of these techniques has been used for great effect, even helping to position chromosomes in interphase nuclei (112), and is now being used with multiple probes and colors, such as in the paper from Cremer’s group, whereby they look at chromosomal regions, gene expression, and histone methylation (126).
Locked nucleic acids (LNAs) are a class of RNA analogs with exceptionally high affinity toward complementary DNA and RNA (127). Because of the LNA chemical makeup, heteroduplexes between LNA oligonucleotides and their complementary DNA oligonucleotides  show a shift in structure from a B-like helix toward an A-type helix. This results in a higher thermal stability of the LNA-DNA heteroduplexes (128). LNA-FISH refers to the use of LNA-modified oligonucleotides in FISH experiments for improved resolution and sensitivity (129,130).
The invention of M-FISH (or Multiplex-FISH), a protocol for 24-color karyotyping, based on combinatorial labeling (131) and aimed at facilitating the analysis of complex chromosomal rearrangements and marker chromosomes, has signified a groundbreaking development in molecular cytogenetics, particularly for the study of tumors and prenatal diagnosis. Dissimilarly from ratio-labeling based multicolor approaches, in which chromosomespecific probes are characteristically labeled with different proportions of fluorochromes and accurate measurements of relative fluorescence intensity are required, the M-FISH technique (like the related SKY technique, see below) consists of labeling each probe with a unique combination of five spectrally separable fluorochromes in a 1:1 ratio. Accordingly, although relying on the use of narrow band-pass fluorescence filters appropriately set in a motorized filter wheel and on digital imaging software more sophisticated than the standard setup for FISH analysis, the interpretation of the results is relatively straightforward (that is, the fluorochrome is either present or absent). The technique was originally devised for use with and simultaneous detection of the 24 human chromosome painting probes (22 autosomes and the X and Y chromosomes), but has been subsequently used to analyze specific chromosomal subregions, like centromeres and subcentromeres in protocol variants for the characterization of small supernumerary marker chromosomes with no euchromatin. Examples include cenM-FISH (132), CM-FISH (133), and subcenM-FISH (134), telomeres for the identification of subtle subtelomeric rearrangements as in M-TEL (135,136), and chromosome arm-specific probes (see armFISH above) for the detection of pericentric inversions. Similar in principle and application to the M-FISH technique is the spectral karyotyping technique or SKY (137), in which chromosomes are classified on the basis of their unique emission spectra. For image acquisition and analysis, SKY requires specific hardware and software, comprising a custom-designed single triple-band-pass filter and an interferometer able to retrieve spectral information for every pixel in a digital image. For comprehensive reviews on 24-color FISH analysis see Kearney et al. (2006) (138) and Schrock et al. (2006) (139). M-FISH is also used as a term to mean Multicolor-FISH.
Multilocus or ML-FISH
The word multilocus (subsequently abbreviated with the acronym ML) refers to the simultaneous use in multicolor FISH of multiple probes. This FISH assay was initially designed to screen for multiple microdeletion syndromes in patients with unexplained developmental delay and/or mental retardation (140). The original multilocus panel designed included Prader-Willi, Angelman, Williams, Di George/velocardiofacial, and Smith-Magenis syndromes. Based on the same principle, a multilocus strategy with locus-specific DNA probes for chromosomes 13 and 21 was more recently applied to the study of nondisjunction in mature human oocytes (141).
PCC-FISH is a FISH application used for biodosimetric analysis that relies on the use of chromosome-specific painting probes to determine chromosome damage after irradiation. The acronym PCC stands for premature chromosome condensation and refers to the effect obtained by virusmediated cell fusion or phosphatase inhibitors (either calyculin A or okadaic acid) to prematurely condense the chromosomes of cells in G1 and G2 phases, a central aspect of the procedure that overcomes the need to culture cells in vitro as required for conventional metaphase chromosome analysis. PCC-FISH was initially devised as an assay to estimate/ predict the in situ radiation sensitivity of individual human tumors (142–144). It has subsequently been used to estimate the effect of whole-body high- or low-dose exposure to human peripheral lymphocytes (145,146), and carried out on skin fibroblasts in the case of acute localized irradiation following accidental overexposure (147). PCC-FISH has also been used to establish the radiosensitizing effect of drugs in experimental cancer treatments (75,148,149). The technique is used additionally in in vitro studies for general radiation research and cancer research purposes.
Bridged nucleic acid 2',4'-BNANC (2'-O,4'-aminoethylene bridged nucleic acid) is a compound containing a six-member bridged structure with an N-O linkage. This novel nucleic acid analogue can be synthesized and incorporated into oligonucleotides. When compared to the earlier generation of LNA, BNA was found to possess:  BNA oligomers to complementary DNA or RNA sequences, and BNA-DNA and BNA-RNA duplexes are more stable than the natural homo- or heteroduplexes. FISH with DNA hybridization is more significantly affected by base mismatches than DNA-DNA hybridization (153) and that BNA probes could discriminate between two centromeric repeats that differed only by a single base pair endorsed this as an important development in the field. Thus, short (15- to 18-mer) BNA probes for a-satellite domains of specific chromosomes were designed, and their power of discrimination at a single-base level was used for unique chromosome identification in metaphase and interphase (155,156). As well as on lymphocytes, amniocytes and fibroblasts, BNA-FISH has also been successfully carried out on human spermatozoa (157) and isolated oocytes, polar bodies, and blastomeres (158), a strong indication of the potential of BNA probes for preimplantation cytogenetic diagnosis. Larsen and collaborators (159) tested the suitability of BNA-FISH for noninvasive prenatal diagnosis by detecting ?-globi?-globin messenger RNA (mRNA) in fetal nucleated red blood cells from maternal blood. Also, as a result of the relative hydrophobic character of BNA compared with DNA, that allows better diffusion through the cell wall, BNA-FISH has also had wide application in microbiology, for both research and clinical purposes (160,161). Because of its high binding specificity quality, BNA technology is expected to provide a platform for the design of allele-specific probes for in situ hybridization, a technical development longed for by many in the genetic, cytogenetic, and epigenetic ranks.
Quantitative-FISH methodology permits the measurement of probe signal intensity. Q-FISH was initially invented by Lansdorp and collaborators (162) and was shown to work in combination with flow cytometry (163) (see above for Flow-FISH). This method has been used mainly for measuring the number of telomere repeats on a particular chromosome, using BNA-conjugated probes. Typically, metaphases are imaged and then analyzed using software such as TFL-TELO (162,164). The lengths of chromosomal regions can be measured at the resolution of 200 bp (165). Q-FISH has advanced and is now used on interphase cells and tissue sections. Indeed, in interphase cells, a similar methodology with a different acronym has been used (i.e., single telomere length analysis or STELA) (166). Q-FISH has also been used to quantify telomere length in interphase cells (167) and has even been given the acronym IQ-FISH for Interphase Q-FISH (168). Q-FISH has become an important tool in studying the role of telomeres in aging and cancer.
QD-FISH refers to the recently pioneered utilization of quantum dot-conjugates in FISH protocols. Quantum dots are nanometer-sized inorganic fluorophores, characterized by photostability and narrow emission spectra, that have been successfully used for FISH analysis on human metaphase chromosomes (169), human sperm cells (170), bacterial cells (171), and also to detect subcellular mRNA distribution in tissue sections (172). While initial QD-FISH protocols entailed hybridization of biotinylated probes and subsequent detection using streptavidin-conjugated QDs, more recent adaptations of the procedure, aimed at multicolor imaging, involve direct labeling of oligonucleotide probes (172,173).
Rainbow-FISH is an advanced digital imaging procedure that allows the simultaneous detection and quantification of up to seven different microbial groups in a microscopic field. The technique was designed to improve quantitative analytical studies of microbial communities from various aquatic environments (174). Based on similar principles to the multi-FISH technique previously developed for the analysis of microbes in human feces (175), the rainbow protocol brings together the use of specific 16S ribosomal RNA (rRNA)-targeted oligonucleotide probes for the discrimination of different phylogenetic groups of microbes (176) and the principles of combinatorial labeling. As a result, by the combined application of seven DNA probes, each labeled with up to three fluorochromes, seven kinds of microbial strains can be distinguished simultaneously (177). The specific Rainbow-FISH digital procedure developed by Sunamura and Maruymama (174) consists of systematic background noise reduction and target signal equalization to discriminate between microbes and nonmicrobial particles, and aims at facilitating image processing and analysis, in particular the normalization steps that are usually very laborious when imaging natural environmental microbes, since their cellular rRNA content can vary between cells, even within the same sample.
Raman-FISH is a new technique that combines FISH technology with Raman microspectroscopy for ecophysiological investigations of complex microbial communities (178). The procedure exploits the so-called red shift phenomenon or the significant change in the resonance spectra, as visualized by Raman microscopy, that follows anabolic incorporation of 13C isotope, when compared with normal 12C, into microbial cells. Metabolic labeling through stableisotope incorporation is combined with microbial species identification by in situ hybridization with specific 16S rRNA probe for structural and functional interrelated analyses of microbial communities at a single-cell resolution.
ReD-FISH, which stands for replicative detargeting FISH, is similar to CO-FISH, whereby BrdU is added to the culture medium during DNA replication for incorporation into the newly synthesized strand. ReD-FISH allows the replication timing of specific sequences to be determined. If BrdU has been incorporated in the sequence of interest, the newly formed DNA strand will be detargeted (as in CO-FISH, see above), and each oligonucleotide probe will only be able to hybridize to one of the parental strands, and only one chromatid will be display a signal. However, if the sequence of interest has not been replicated and not incorporated BrdU, then a FISH analysis will reveal the standard double signal on both chromatids of the metaphase chromosome. The ReD-FISH technique has been instrumental in ascertaining the replication timing of telomeres, leading to the observation that telomeres replicate throughout S-phase and that telomeres from p and q arms of the same chromosome replicate asynchronously (179).
First demonstrated in 1990 (180), Reverse-FISH is the process whereby the FISH probe comprises DNA from the material of interest. This can be a chromosome of a specific species in the cellular background of another species (i.e., a somatic cell hybrid). It can also be chromosomal material obtained in other ways such as by flow sorting (181) or microdissection (182,183). The reverse terminology refers to the probe being the material of interest, usually aberrant, and being painted onto control or reference metaphase chromosomes to identify what sequences/chromosomal regions the probe contains or is missing. Reverse-FISH has been useful for characterizing marker chromosomes (184–186) and chromosome amplifications in cancer (187).
In situ hybridization of oligonucleotide probes to high copy number nucleic acid targets, such as rRNA, is a standard method for the identification of micro organisms in enviromental samples. RING-FISH is a modified version of this technique that relies on the use of high concentrations of polynucleotide probes for an increase in sensitivity and visualization of any part of the genetic material of a bacterial cell, regardless of copy number (188,189). The signal amplification achieved with these polynucleotides, the length of which ranges between one to several hundred base pairs, is mediated by their secondary structures and intermolecule interactions, resulting in a conspicuous network of polynucleotides that builds upon the initial hybridization site. RING stands for recognition of individual genes, but also refers to the characteristic ring-shaped or halo appearance of the fluorescence signals at the bacterial cell periphery (188).
RNA-FISH is a method that allows detection of RNA within cells. Transcripts can be visualized either in the nucleus or in the cytoplasm. The technique, also known as expression-FISH, has been used to analyze the transcriptional activity of endogenous genes (190) as well as exogenous genes such as those belonging to integrated viral genomes (191,192) and transgenes (193). The technique permits investigation into allelic-specific expression on a per cell basis (194,195) and is expected to provide a platform for gene expression profiling studies in single cells (196). RNA-FISH has also been instrumental in studying different functional aspects of genome organization and nuclear architecture (124,197– 205). Further, as a technique, it is being examined as a prenatal diagnosis tool for myotonic dystrophy type 1 (206).
RxFISH is a color banding technique that is also described as chromosome bar-coding (207). The method relies on sequence homologies between human and the apes, such as gibbon (98%), to produce, by cross-species hybridization, a specific banding pattern on human metaphase chromosomes (208–210). This is possible because of the intrachromosomal rearrangements that have occurred during primate evolution. If the probes are labeled with a number of fluorochromes, usually three, this allows a colorful and reproducible banding to be observed and analyzed. Due to the color bands, it is easier than G-banding to see chromosomal rearrangements, especially intrachromosomal rearrangements (211). However, in combination with G-banding, RxFISH can provide very detailed information about chromosomal breakpoints, for example in cancer (212–214).
Split-Signal FISH
Split-Signal FISH is a fast and sensitive dual-color FISH assay for the detection of frequently occurring chromosome translocations affecting specific genes in hematopoietic malignancies. The technique is equally suitable for metaphase and interphase analysis and has been increasingly used for both diagnostic and prognostic purposes. In its current form, the assay involves the design and differential labeling of two probes from the flanking regions of the translocation breakpoint. The signals normally co-localize and appear fused, but as a result of the translocative event, they will split. Split-Signal FISH was initially introduced as an innovative and simple experimental approach for the detection of all types of MLL gene translocations gene translocations in ALL and AML, using only a single FISH test (215). However, a prototypic version of the protocol with differentially labeled probes for genes spanning or adjacent to the translocation breakpoints, on the two chromosomes involved, had been previously used for the detection of the Burkitt translocation t(8;14) in B cell lymphomas (216) and the detection of t(11;14) in mantle cell lymphomas (217). See also related Fusion-Signal FISH technique.
The T in T-FISH can stand for tyramide, tissue, or telomere. The three versions of T-FISH are discussed in the order of their arrival in the field. Tyramide-FISH: tyramide is a compound that binds to peroxidase easily and thus has been used to increase the sensitivity greatly in FISH experiments, with the use of only one or two layers of reagents for visualization (218). The first layer uses a peroxidase-conjugated antihapten antibody or a compound such as strepavidin to bind to the labeled probe. Fluorochromes or haptens, such as biotin, are conjugated to tyramine derivatives. This leads to a massive build-up or towers of fluorochromes or moieties that can be visualized by fluorochromes, making detection ultrasensitive (219). The chemistry behind tyramide signal amplification (TSA) is now licensed, and kits can be purchased from various companies. The technology has been used to map gene loci and look for specific transcripts in cells (220–222). Tissue-FISH: tissue samples are collected frequently from patients or experimental animals. These samples can be frozen, fixed, or embedded in paraffin wax. A number of research groups have developed methods of delineating sequences in tissues by FISH, for example, in paraffin sections and frozen sections (223). However, it was Nomura and collaborators (224), who, in 2003, coined the term T-FISH to denote FISH on tissue sections. Telomere-FISH: some groups have called FISH using telomeric probes T-FISH (225–227).
3-D FISH was developed in Germany by the groups of Peter Lichter and Thomas Cremer (124,228,229) to analyze spatial positioning and relative organization of chromosomes and subchromosomal regions within cell nuclei. The methodology relies on using cross-linking fixation reagents, such as paraformaldehyde, to preserve nuclear architecture and large-scale chromatin organization (230,231). Due to the resulting crosslinking of proteins, an effective permeabilization step is required to allow the probes to penetrate the sample. This is often performed using detergents and freeze and thaw cycles at -180°C. To visualize the 3-D signals, operators will need to use confocal laser-scanning microscopyor use accurate deconvolution tools. One of the most significant achievements in the field was the publication of a paper displaying the simultaneous painting of all human chromosomes in the nuclei of three-dimensionally preserved cells (232). The introduction of 3-D FISH protocols and their wide application in the field of chromosome biology has significantly propelled, over the last few years, our understanding of chromosome structure and function.
Zoo-FISH, also known as crossspecies chromosome painting, consists of hybridizing libraries of DNA sequences, also known as chromosome paints, from one species to the chromosomes of another species, to identify regions of synteny. From a methodological point of view, the protocol does not differ significantly from standard FISH protocols, but there can be some issues with signal intensity and background noise, as pointed out by Solovei (www.biologie.uni-muenchen.de/ou/humbio/pdf/solovei/protocol/11_zoofish.pdf). Solovei discusses that good metaphase spreads are absolutely fundamental, that suppression of repetitve sequences may be unnecessary, and that higher concentrations of probe are usually required. A critical step to be borne in mind is that the more divergent the organisms are from each other, the lower the stringency should be within the protocol. The first study using Zoo-FISH used human and mouse whole chromosome painting probes on primates, rodents, even-toed ungulates, and whales (233). Since then, this type of chromosomal analysis has been widely used, permitting the establishment of important chromosome homology maps, revealing interesting insights into chromosome rearrangements during evolution, and allowing us to infer information on ancestral karyotypes. The impact of Zoo-FISH on the comparative study of genome evolution has been reviewed in different papers (234–237).
The development of such an abundance of exciting FISH protocol variants and applications that we report and discuss in this glossary is the result of numerous, generally independent, but also collaborative efforts of many different laboratories in the world. Due to space limitations we have been unable to cite all relevant/significant papers, and we would like to apologize to friends and colleagues whose contribution to the field has been unwillingly omitted.
The authors declare no competing interests.
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Received 22 October 2007; accepted 30 January 2008.
Address correspondence to Emanuela V. Volpi, Molecular Cytogenetics Laboratory, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. e-mail: evolpi@well. ox.ac.uk and/or Joanna M. Bridger, Laboratory of Nuclear and Genomic Health, Centre for Cell and Chromosome Biology, Division of Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge, UK. e-mail: joanna.bridger@brunel.ac.uk