Tyramide Signal Amplification: New Opportunities for DNA <i>In Situ</i> Hybridization

Abstract

DNA in situ hybridization (DNA-ISH) is a widely used method in molecular cytogenetics that allows the localization of specific DNA sequences in particular regions of chromosomes. Implementation of DNA-ISH requires the use of DNA probes, which can be commercial or developed for specific research purposes as non-commercial (homemade) DNA probes. One of the significant drawbacks of non-commercial probes is the difficulty in obtaining a high signal intensity with a small DNA probe size. Therefore, developing approaches to enhance non-commercial DNA probes is an important task in modern molecular cytogenetics. To directly visualize small DNA sequences on a chromosome, the tyramide signal amplification (TSA) method is used. The TSA system is based on the formation of a covalent bond between electron-rich protein fragments in the sample and tyramide molecules linked to a hapten (in chromogenic in situ hybridization) or a fluorophore (in fluorescent in situ hybridization). This is achieved by converting tyramide molecules into free-radical intermediate compounds under the action of horseradish peroxidase (HRP), followed by deposition of precipitated molecules nearby. As a result, a low-intensity signal is amplified. Thus, TSA is a good complement to the DNA-ISH method, thanks to its high sensitivity and ability to detect small genomic imbalances, and can therefore become a valuable tool for diagnosing chromosomal rearrangements in clinical practice.