CLONING OF RNA MOLECULES INVITRO

A method for RNA amplification in an immobilized medium is described. The medium contains a complete set of nucleotide substrates and purifi ed Qbeta replicase, an enzyme capable of exponentially amplifying RNAs under isothermal conditions. RNA amplification in the immobilized med ium results in the formation of separate 'colonies', each comprising t he progeny of a single RNA molecule (a clone). The colonies were visua lized by staining with ethidium bromide, by utilizing radioactive subs trates, and by hybridization with sequence-specific labeled probes. Th e number and identity of the RNA colonies corresponded to that of the RNAs seeded. When a mixture of different RNA species was seeded, these species were found in different colonies. Possible implementations of this technique include a search for recombinant RNAs, very sensitive nucleic acid diagnostics, and gene cloning in vitro.