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Shall you use Biotin or Digoxigenin for ISH Probe Labeling?

Posted By Michael Yan
Tags: Successful Research Tips

ISH, or in situ hybridization, is a powerful technique designed to detect specific nucleic acid sequences within cells and tissue samples. The technique is based on the hybridization between complimentary DNA or RNA probes to a particular target sequence. It is mainly used to detect the presence of specific genomic sequences of any type, including bacteria and viruses. Detection enables the determination of spatial location of sequences within tissues and cells, while also providing insight on relative levels of expression. When stored in optimal conditions, tissue samples can be maintained with minimal signal degradation for years.

The first generation of probes used for ISH were introduced in 1969. These probes were based on radiolabeling technologies, which frequently used isotopes such as 23P, 35S, and 33P. These radioisotopes initially showed promising results displaying high activity and visualization via photographic film exposure. However, these methods of detection were unfortunately hindered by various constraints that reduced practicality and convenience. Isotopic probes are inherently unstable due to the relatively short half-lives of their associated isotope labels. This means that tissue samples are usually short lived and cannot be maintained for extended periods of time. Even more concerning is the potential hazards implicated on end users due to radioactive exposure, necessitating more stringent protection.

Because of these potential hazards and drawbacks, developments were made to create alternative options such as non-isotopic probes using other haptens. Two common non-isotopic haptens used to generate ISH probes are biotin and digoxigenin. Detection mechanisms typically utilize fluorescence or enzymatic colorimetric development through antigen-antibody interaction in the case of digoxigenin and streptavidin affinity binding to biotin. Methods of creating non-isotopic probes usually require incorporation of conjugated UTP into sequences. This can be conducted through nick translation or random priming. While direct conjugation of probes using Alkaline phosphatase (AP) or fluorophores is possible and has existed, it is not used as frequently due to expensive costs. Overall, non-isotopic haptens are known to have better resolution, stability, and shorter development times compared to their isotopic counterparts.

The first non-isotopic hapten to find widespread success were biotin-conjugated ISH probes. Biotinylated probes are further enhanced by higher affinity binding to streptavidin, which is usually conjugated with alkaline phosphatase for color development. Biotinylated probes are usually easier to work with and are less expensive to generate in contrast to isotopic variants. Because radioactive decay is not a factor, biotinylated probes can be retained in tissue samples for extended periods of time as long as they are stored in appropriate conditions protected from light. Despite this, there are several known inefficiencies unique to biotinylated systems. Traditionally, biotin could be incorporated through direct in-vitro transcriptional reactions via biotin-coupled rNTPs. Alternatively, incorporation can also be made through allylamino-UTP with subsequent coupling of the allylamino group to biotin-N-hydroxysuccinimide. However, both processes have shown notable differences in transcription rates due to steric hindrances of conjugated molecules, resulting in suboptimal nucleotide interaction with transcriptional machinery. It is also known that tissue samples contain endogenous levels of biotin. Since the conjugated streptavidin associates with biotin at a high affinity, the possibility for producing high background and false positives exists.



Figure 1: Workflow of hybridization with DIGX® HPV Probes and detection with DIGX® linkers and POLYVIEW® PLUS reagents.
In response to the problems seen in biotinylated systems, digoxigenin-conjugated systems have been investigated as a potentially better alternative. Digoxigenin is derived from the flowers and leaves of the Digitalis Purpurea plant. It is an endogenous steroid found exclusively in these plants and will not appear in any animal cell or tissue samples. This circumvents the previously mentioned background staining with biotin as there is increased specificity for associated conjugate-antibodies to target sequences. Since the detection mechanisms involve antibody sandwich mechanisms, it allows for more conjugated-antibody fragments to bind onto a single digoxigenin molecule and generates higher signal amplification.


Figure 2: DIGX® HPV type 16/18/31/33/51 probe (ENZ-GEN114) was used to assay cervical tissue using a Leica Bond III. Image was acquired with 10x objective.
Enzo Life Sciences offers a complete set of solutions for in situ hybridization, providing everything you need for labeling, hybridization, and detection. Our DIGX® HPV probes and linkers are available in specific HPV genoptypes and can adapt DIGX probes to antibody-based detection systems, such as our POLYVIEW® PLUS kits. For a simple and effective method of generating labeled DNA, our Nick Translation DNA Labeling System 2.0 provides all the necessary reagents required and can accommodate a wide range of fluorophore-labeled, biotin-labeled, and digoxigenin-labeled nucleotides. For additional information on ISH or any of our other product lines, please see our brochures. Please contact our Technical Support Team for questions on any of our products.

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