Cloning of RNA molecules in vitro.

Nucleic acids research ·Vol. 21 ·No. 10 ·1993-05-25 ·Pages 2349-53

Chetverina HV, Chetverin AB

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.

MeSH Terms

Cloning, Molecular Ethidium Gene Amplification In Situ Hybridization Nucleotides Q beta Replicase/metabolism RNA/genetics RNA Probes Staining and Labeling Templates, Genetic

Chemicals

Nucleotides RNA Probes RNA Q beta Replicase Ethidium

Authors & Affiliations

2 authors, click to expand affiliations / ORCID

Chetverina H V

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region.

Chetverin A B

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Article Info

Journal

Nucleic acids research

Abbr.

Nucleic Acids Res

ISSN

0305-1048

DOI

10.1093/nar/21.10.2349

Published

1993-05-25

Pages

2349-53

Language

English

Region

England

NLM ID

0411011

PMCID

PMC309531

Subset

External Links

PubMed source DOI: 10.1093/nar/21.10.2349 PMC Full Text View journal details

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Cloning of RNA molecules in vitro.

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