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What methods are used for probe generation and detection in FISH and CISH?

Posted By Jan-Henning Martens
Tags: Molecular Biology, Successful Research Tips

In situ hybridization (ISH)

In situ hybridization (ISH) is a unique molecular analysis method providing accurate localization of endogenous, bacterial or viral nucleic acids such as DNA, mRNA, and microRNA in metaphase spreads, cells, and tissue preparations. Fixation and pre-treatment of samples are followed by hybridization of a labeled probe by complementary base pairing to the target and detection of the label to allow microscopic visualization of the hybrid. Detection of the probe can be achieved by chromogenic or fluorogenic techniques referred to as chromogenic in situ hybridization (CISH) or fluorescent in situ hybridization (FISH), respectively. One common application is the detection of genetic aberrations in cancer and in prenatal/postnatal samples.


ISH probe generation techniques

Because hybridization can take place between complementary deoxyribonucleotides or ribonucleotides, either DNA- or RNA-based probes (riboprobes) can be used to localize DNA or RNA in a given sample. Commercially available probes are expensive and do not always offer experimental flexibility as they come with a single label type. Therefore, many researchers still heavily rely on preparing their own FISH probes by using one of the following techniques: nick translation or random-primed labeling for generation of long, double-stranded DNA probes, terminal-labeling of oligonucleotides and in vitro transcription from vectors containing RNA polymerase promotors to produce riboprobes.
CISH and FISH probes for detection of nucleic acids in human as well as non-human samples are usually generated from BAC clones. They can be prepared by random-primed labeling with the use of DNA polymerase and labeled deoxyribonucleoside triphosphates (dNTPs) to lengthen random oligonucleotides that hybridize to DNA sequences of the denatured vector. Alternatively, they may be prepared by nick translation which is often the preferred method of choice. In nick translation, the DNA to be labeled is nicked by DNase I, yielding a free 3′ hydroxyl end. DNA polymerase I then adds a new nucleotide to this end. The 5′-3′ exonuclease activity of the polymerase then moves the “nick” along the strand in the 3′ direction, and the addition of a labeled nucleotide to the reaction results in the desired probe. We recently launched our Nick Translation DNA labeling system 2.0 kit to provide a simple, rapid, reliable, and efficient method for the end users to generate their own labeled DNA probes for both CISH and FISH in just one hour. A ready-to-use nick translation enzyme mix that has an optimized concentration of DNA polymerase I and DNase I, is included in the kit to simplify the protocol. This kit can be used with separately accessible dNTPs labeled with biotin, digoxigenin, as well as fluorophores. Additionally, the kit design allows the user to enhance label incorporation by adjusting the ratio of labeled-dUTP and dTTP, and to optimize reaction product size by varying the time of DNase I digestion.
For many years, automatic DNA synthesizers have easily chemically synthesized oligonucleotide DNA probes 20 to 50 bases long that are specific to any target DNA sequence of interest. These oligonucleotides can either be directly labeled during their synthesis or labeled nucleotides may be added to their ends using terminal deoxynucleotidyl transferase or T4 polynucleotide kinase.
Although RNA probes can be very problematic to work with, RNA ISH using riboprobes has been gaining traction lately. Riboprobes are prepared by either using a specially designed RNA expression vector or by attaching RNA polymerase recognition sites to vectors containing the probe sequence of choice. During in vitro transcription in the presence of labeled and unlabeled ribonucleotides, RNA polymerase is able to generate single-stranded riboprobes from the DNA template.


ISH probe detection techniques

Direct detection is possible thanks to fluorescent labels that can be introduced during FISH probe synthesis and detected by fluorescence microscopy. Multiplexing can easily be envisaged as two or more different probes labeled with different fluorophores can be visualized at any single time. Enzo offers fluorophores with several distinct colors to choose from, spanning the visible light spectrum and guaranteeing high signal intensity and good photostability. This direct approach provides a simple and efficient method to label multiple sequences for FISH. Multiplex DNA FISH can be used to identify genetic rearrangements (e.g. ALK, BCR-ABL, HER2, MYB) while multiplex RNA FISH allows the generation of expression profiles, both in time and in space, of several mRNA targets.
Indirect detection constitutes a valid alternative as it relies on an intermediary and a chromogen to produce staining that can endure the stress of time, much like immunohistochemistry (IHC). Biotin is commonly used in indirect systems to label oligonucleotide probes. Biotin can then be detected with avidin from egg white or streptavidin from the bacteria S. avidinii, proteins known to have a high binding affinity to biotin. These proteins are conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP), which can react with specific substrates to produce a chromogenic precipitate for CISH. One critical pitfall is that biotin is endogenously expressed in cells and tissues leading to nonspecific staining. Digoxigenin is a non-radioactive immune tag isolated from the Digitalis plant and as such, is unlikely to bind to other biological material. It can be paired with anti-digoxigenin antibodies to allow the detection of oligonucleotides of interest with high affinity, sensitivity, and most importantly, specificity. Digoxigenin-based detection overcomes high background or false positives that may arise from endogenous biotin in biological samples when using biotin-based detection. Thanks to the availability of a panel of chromogens of different colors (e.g. black, blue, brown, green, red, and yellow) and increased flexibility with AP and HRP options, sequential ISH-ISH or ISH-IHC in the same tissue sections can be developed and optimized to facilitate correlations between different markers. For example, HPV DNA probes are used to detect and identify human papillomavirus by ISH in head and neck cancers. P16, a protein involved in the control of the cell cycle, is often used as a surrogate marker of HPV DNA detection for both prognosis and therapeutic purposes, hence the relevance of trying to combine HPV DNA/RNA-ISH with p16-IHC on a single tissue section.

Enzo offers a complete set of solutions for labeling, hybridization, and detection. Check out our Genomics platform for more information and our Successful Research Tips. Additionally, contact our Technical Support Team for further assistance.

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