Open AccessCloning of RNA moleculesin vitro
Author(s) -
Helena V. Chetverina,
Alexander B. Chetverin
Publication year - 1993
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/21.10.2349
Subject(s) - biology , rna , ethidium bromide , microbiology and biotechnology , rna dependent rna polymerase , nucleic acid , cloning (programming) , recombinant dna , molecular cloning , nuclease protection assay , clone (java method) , in vitro , complementary dna , biochemistry , gene , dna , computer science , programming language
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.