Identifying RNA recombination events and non-covalent RNA-RNA interactions with the molecular colony technique.

Abstract

Molecular colonies (also known under names nanocolonies, polonies, RNA or DNA colonies, PCR colonies) form when nucleic acids are amplified in a porous solid or semi-solid medium, such as a gel, which contains a system for the exponential multiplication of RNA or DNA. As an individual colony comprises many copies of a single molecule (a molecular clone), the method can be used for the detection, enumeration, and analysis of individual DNA or RNA molecules, including the products of such rare events as RNA recombinations. Here we describe protocols for the detection of RNA molecules by growing colonies of RNA (in a gel containing Qβ replicase, the RNA-dependent RNA polymerase of phage Qβ) or cDNA (in a gel containing the components of PCR), and visualizing them by hybridization with fluorescent probes directly in the gel, including in real time, or by hybridization with fluorescent or radioactive probes followed by transfer to a nylon membrane.