Biomerieux, Inc.

William McAllister

Bernard F Mandrand

Guy Oriol

Philippe L Cleuziat

François Mallet

Pierre Levasseur

Francedillaois Mallet

Fran&ccedil;ois Mallet

Nadège Arnaud-Barbe

Val&eacute;rie Cheynet-Sauvion

Francedillaoise Guillou-Bonnici

Valérie Cheynet-Sauvion

Nad&eacute;ge Arnaud-Barbe

RNA may be transcribed using a nucleotide reagent as the promoter. The reagent may enable RNA to be transcribed without sequence specification and without protein cofactors, by means of an RNA polymerase that is known to be DNA-dependent such as the RNA polymerase of the phage T7, or by means of new, mutated RNA polymerase with the ability to synthesize a transcription product of polynucleotide matrix with a higher yield when the matrix is RNA than when the matrix is DNA. This type of RNA polymerase can be obtained by effecting mutations on a coding gene for a wild-type RNA polymerase, and then by selecting the mutated RNA polymerase with the ability. The invention can be applied notably to the detection, synthesis or quantification of RNA.

RNA-dependent RNA polymerase functioning preferably on a RNA matrix and promoter-dependent transcription process with said RNA-dependent RNA polymerase

Disclosed is a process for transcribing RNA using a nucleotide reagent as the promoter. Such a reagent enables any type of RNA to be transcribed without sequence specification and without protein cofactors, by means of an RNA polymerase that is known to be DNA-dependent such as the RNA polymerase of the phage T7, or by means of new, mutated RNA polymerase with the ability to synthesize a transcription product of a polynucleotide matrix with a higher yield when the matrix is RNA than when said matrix is DNA. This type of RNA polymerase can be obtained by effecting mutations on a coding gene for a wild-type RNA polymerase, and then by selecting the mutated RNA polymerase with said ability. The invention can be applied notably to the detection, synthesis or quantification of RNA.

Method of amplifying an RNA target sequence using an RNA polymerase functioning preferably on RNA template

A chimera oligonucleotide is provided that can be used in a process for obtaining transcripts and/or amplification of a target sequence of a nucleic acid, having, at its 3' end, a downstream sequence. The oligonucleotide comprises successively, from 5' to 3', 1) a first oligonucleotide segment, of the DNA type, comprising a sense sequence of a promoter of an RNA polymerase, 2) a second oligonucleotide segment, of the DNA type, capable of hybridizing with the downstream sequence, and 3) a third oligonucleotide segment, of the RNA type, capable of hybridizing with a part of the target sequence contiguous to the downstream sequence, the third segment being blocked at 3'. A process using the chimera oligonucleotide and an enzyme system containing DNA polymerase activity, RNA polymerase activity, and a third activity, for example, an RNase H activity provides transcription products of the target. By adding a second chimera oligonucleotide capable of hybridizing with the complement of the target, cyclic amplification of the target and its complement are obtained.

Chimera oligonucleotide and its utilization for obtaining transcripts of

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Perth Amboy, NJ, United States of America

William McAllister