TARGET AND SIGNAL AMPLIFICATION - APPROACHES TO INCREASE THE SENSITIVITY OF IN-SITU HYBRIDIZATION

In situ hybridization (ISH) has proven to be a very important molecula r tool in research and diagnosis. However, its applicability can be li mited by its restricted detection sensitivity. During the last few yea rs, several strategies have been developed to improve the threshold le vels for ISH detection by amplification of either target nucleic acid sequences prior to ISH or the detection signals after hybridization pr ocedures. In this overview, we outline and analyze the principles, app lications, and limitations of in situ polymerase chain reaction, in si tu self-sustained sequence replication, primed in situ labeling (PRINS ), and in situ transcription as examples of target amplification metho ds, and catalyzed reporter deposition using biotinylated tyramine as a n approach to signal amplification in ISH. We also provide a detailed, 1-day protocol for non-radioactive oligonucleotide ISH including sign al amplification, which is suitable for diagnostic purposes. Furthermo re, future directions of ISH including combined strategies of target a nd signal amplification as well as automation are discussed.