Cloning of RNA molecules in vitro.
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.
Chetverina
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HV
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region.
Chetverin
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AB
D003001:Cloning, Molecular; D004996:Ethidium; D005784:Gene Amplification; D017403:In Situ Hybridization; D009711:Nucleotides; D011777:Q beta Replicase / Q000378:metabolism; D012313:RNA / Q000235:genetics*; D015347:RNA Probes; D013194:Staining and Labeling; D013698:Templates, Genetic
D009711:Nucleotides; D015347:RNA Probes; D012313:RNA; D011777:Q beta Replicase; D004996:Ethidium