Summary

This protocol is a prototype for a series of upcoming procedures aimed at the detection of single molecules of nucleic acids in situ. It uses circular probes that, upon hybridization to their target molecule, can template rolling-circle DNA synthesis, if the target also provides a primer for initiation of the DNA synthesis. This primer may be endogenous or artificial, for example, obtained through cleavage with a restriction enzyme. In this format, the primed in situ labeling (PRINS) product takes the form of a long-tandem repeat copy of the probe, covalently attached to the site of synthesis, and tagged it with copies of the probe. The amplification is sufficient for the detection of single, oligonucle-otide size, targets and, depending on the probe format, the probe may detect variations in the targets down to single base substitutions.

Key Words: PRINS; RCA; rolling circle. 1. Introduction

Organisms are made of tissues, and tissues are made of cells. Any tissue is composed of a mixture of many types of cells. On a genetic level, these cells differ in their gene expression and, in cancers, also in their DNA. Because the performance of any tissue and, ultimately, the organism, depends on the behavior of its constituent cells, a thorough understanding of biological processes in biology and in pathology requires studies of the individual cells and the biomolecules within the cells. The dominant molecular biology methods do not provide this insight because they rely on the analysis of biomolecules isolated from extracts of pools of cells. To get the full picture, it is necessary to obtain data on the individual cells in a format that also gives spatial information on the biomolecules inside the cells and the cells inside the tissues. In essence, this means that cells and biomolecules should be studied in situ, which

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed. Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

has been possible since the introduction of in situ hybridization and immuno-histochemistry. Unfortunately, any of the two techniques has a limited sensitivity (cannot see ultimately low amounts of targets) and resolution (cannot see ultimately small features of the targets). I therefore undertook the development of a new technology with the prospect of providing single-molecule DNA and RNA detection in situ at single-nucleotide resolution approx 20 yr ago (1-4). The underlying principles of the new technology were that short oligonucle-otides were used as probes for the target definition and DNA synthesis at sites binding the probes was used for target visualization. With oligonucleotide probes being sensitive down to single base variations in the target sequence, it should be possible to identify point mutations (or single-nucleotide polymorphisms) in target sequences and, with the DNA synthesis being capable of proceeding for tens of kilobases in vitro, it should be possible to generate enough DNA from a single priming event for it to become visible in the microscope. This potential has now been turned into a series of protocols, as evidenced by this book. In most protocols, a number of priming events per target are required to make it visible above background because of the low and variable frequency of priming events from endogenous 3'-ends of DNA, either generated during the life of the cell or as a consequence of the cell preparation. Thus, whereas repeated targets enables multiple priming events from a single probe, standard detection of single copy genes has required the use of a cocktail of probes for that gene (5,6). However, there are two ways in which to obtain probe-specific in situ DNA synthesis in the absence of endogenous DNA synthesis, one of which is the rolling-circle PRINS (Fig. 1) described here. The other is the dideoxy-PRINS (see Chapter 8).

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