Thyramide Signal Amplification: New Opportunities for DNA in Situ Hybridization

Abstract—DNA in situ hybridization (ISH) is a valuable technique in molecular cytogenetics for precisely localizing specific DNA sequences on chromosomes. To perform ISH, DNA probes are essential, which can be either commercially available or custom-designed for specific research purposes (homemade probes). However, a drawback of homemade probes is their reduced hybridization signal intensity when they are small. Therefore, it is crucial to develop approaches that optimize the noise/signal ratio when using these DNA probes, which is a current focus in molecular cytogenetics. Tyramide signal amplification (TSA) is a technique that addresses this issue by enabling the visualization of small DNA sequences directly on chromosomes. The TSA system relies on the formation of a covalent bond between electron-rich fragments of sample proteins and tyramide molecules that are linked to a hapten (in chromogenic ISH) or a fluorophore (in fluorescent ISH). This process involves the conversion of tyramide molecules into free radical intermediates by horseradish peroxidase (HRP), followed by the deposition of precipitated molecules in close proximity. As a result, the low-intensity signal is amplified, enhancing the detection sensitivity. TSA serves as an excellent complement to DNA hybridization in situ due to its high sensitivity, allowing the detection of small genomic imbalances. Therefore, it has the potential to be a valuable tool in diagnosing chromosomal rearrangements in clinical practice.