Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification |
| |
Authors: | Mats Nilsson |
| |
Affiliation: | (1) Department of Genetics and Pathology, The Rudbeck Laboratory, 751 85 Uppsala, Sweden |
| |
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.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. |
| |
Keywords: | Padlock probes Rolling-circle amplification In situ genotyping Fluorescence in situ hybridization DNA ligation |
本文献已被 PubMed SpringerLink 等数据库收录! |
|