Recent Developments in Signal Amplification Methods for In Situ Hybridization

In situ hybridization (ISH) allows for the histologic and cytologic localization of DNA and RNA targets. However, the application of ISH techniques can be limited by their inability to detect targets with low copies of DNA and RNA. During the last few years, several strategies have been developed to improve the sensitivity of ISH by amplification of either target nucleic acid sequences prior to ISH or signal detection after the hybridization is completed. Current approaches involving target amplification (in situ PCR, primed labeling, self-sustained sequence replication), signal amplification (tyramide signal amplification, branched DNA amplification), and probe amplification (padlock probes and rolling circle amplification) are reviewed with emphasis on their applications to bright field microscopy. More recent developments such as molecular beacons and in situ strand displacement amplification continue to increase the sensitivity of in situ hybridization methods. Application of some of these techniques has extended the utility of ISH in diagnostic pathology and in research because of the ability to detect targets with low copy numbers of DNA and RNA.