Cloning of RNA moleculesin vitro

Abstract

A method for RNA amplification in an immobilized medium is described. The medium contains a complete set of nucleotide substrates and purified Q beta replicase, an enzyme capable of exponentially amplifying RNAs under isothermal conditions. RNA amplification in the immobilized medium results in the formation of separate 'colonies', each comprising the progeny of a single RNA molecule (a clone). The colonies were visualized by staining with ethidium bromide, by utilizing radioactive substrates, and by hybridization with sequence-specific labeled probes. The 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.

Inorganic Polyphosphate Modulates TRPM8 Channels

“…Chetverin and co-workers have introduced and studied RNA colonies that grew and propagated on gels or other solid media provided that RNA replicases and ribonucleoside triphosphates were present [351,377,378]. It has been explicitly noted that such experimental systems might, in fact, model the amplification and propagation of the first replicators in primordial environmental settings [57,351,354].…”

Qβ Replicase Discriminates between Legitimate and Illegitimate Templates by Having Different Mechanisms of Initiation

“…Amplification of small RQ RNAs is much faster: up to 10 10 copies are produced at room temperature within 10 min in a cell-free system comprised of purified Q␤ replicase and all four NTPs (3), and this is the absolute record of the rate of nucleic acid amplification. The high amplification rate allows single RQ RNA molecules to rapidly produce detectable molecular colonies if they are amplified in a gel (9,10).…”

“…Selection experiments indicated that only a few of the initial diversity of 10 12 unique sequences of 50 -77 nt in length are replicable (4), demonstrating a very high degree of template specificity of the enzyme. Now, almost 40 years since its discovery (11), this specificity remains a mystery.…”

Fluorescent in situ sequencing on polymerase colonies

“…Because acrylamide restricts the diffusion of the PCR amplification products, each single molecule included in the reaction produces a spherical colony of DNA, which we have termed polony (short for polymerase colony). A similar technology, the molecular colony technique, grows nucleic acid colonies in acrylamide using Q beta replicase or PCR [16,17]. Here we describe our progress in developing a protocol for sequencing polonies in parallel by performing repeated cycles of primer extension with fluorescent deoxynucleotides.…”