Abstract
In this review I will describe the development of a technique that enables genotyping of individual DNA molecules in the context of morphologically preserved fixed cells, from the fundamental concept published in 1994 to the present status. The review describes enzyme-assisted histochemistry approaches to achieve highly specific molecular identification reactions coupled to efficient signal amplification. The primary molecular identification is accomplished through circularization of oligonucleotide probes, called padlock probes. The circularization reaction is catalyzed by a DNA ligase, which provides robust distinction between single-nucleotide variants under standard reaction conditions. To generate a detectable signal from individual circularized probe molecules, a DNA polymerase is added that replicates probe circles, generating a long tandem-repeated DNA product, easily visualized using a standard epi-fluorescence microscope. Individual signals are recorded as bright dots, providing digital information about the abundance of specific sequences and opportunities for simultaneous detection of several targets using spectral multiplexing. The importance of strictly target-dependent signal amplification will be discussed.
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Acknowledgments
The results I have described have been achieved through the work of many collaborators and co-workers. I would particularly like to emphasize the contributions of Ulf Landegren, Jörn Koch and Ton Raap, as well as the former and present students Johan Banér, Dan-Oscar Antson, Chatarina Larsson and Sara Henriksson. I further thank Landegren and Raap for valuable comments about the manuscript. My work has been supported by grants from the Biejer, Linné and Wallenberg foundations, from the Swedish Research Council, the EU FP6 integrated project MolTools and by a long-term EMBO fellowship.
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Robert Feulgen Prize 2006 Winner lecture presented at the 48th Symposium of the Society for Histochemistry in Stresa, Lake Maggiore, Italy, 7–10 September 2006.
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Nilsson, M. Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification. Histochem Cell Biol 126, 159–164 (2006). https://doi.org/10.1007/s00418-006-0213-2
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DOI: https://doi.org/10.1007/s00418-006-0213-2