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AssigneeBio Merieux
InventorsVALERIE CHEYNET-SAUVION, NADEGE ARNAUD-BARBE, GUY ORIOL, WILLIAM MCALLISTER, BERNARD MANDRAND, FRANCOIS MALLET
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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 polvmerase can be obtained by effecting mutations on a coding gene for a wild-type RNA polvmerase and then by selecting the mutated RNA polymerase with said abilitv The invention can be applied notably to the detection, synthesis or quantification of RNA.
Method of amplifying any RNA target sequence, by transcription under the control of a promoter, in an RNA sample comprising said target sequence, in which said sample is brought into contact:
Method according to any one of the preceding claims, in which said first and third segments consist of DNA.
Method according to any one of the preceding claims, in which said fourth segment consists of DNA.
Method according to any one of the preceding claims, in which said RNA polymerase is a virus or phage wild-type RNA polymerase.
Method according to any one of the preceding claims, in which said enzyme system contains only an RNA polymerase activity.
RNA polymerase which can be used in the method of any one of the preceding claims, capable of transcribing, under the control of a promoter, a polynucleotide target of interest of any sequence contained in a polynucleotide template, by synthesizing, in the presence of said template and in the absence of associated protein factor, a product of transcription containing an RNA sequence complementary to said sequence, said RNA polymerase being capable of synthesizing said product of transcription with a better yield when said target sequence of said template consists of RNA than when it consists of DNA.
RNA polymerase according to the preceding claim, in which the ratio of the yield of product of transcription of the RNA template to the yield of product of transcription of the DNA template is greater than 2 and in particular greater than 10.
Expression vector into which a gene as defined in the preceding claim is inserted, said vector being capable of expressing said RNA polymerase in a host cell.
Host cell containing an expression vector as defined in the preceding claim.
Use of an RNA polymerase capable of transcribing an RNA template, under the control of a promoter, in the absence of auxiliary protein factor, in a method of transcription of a template strand comprising an RNA target sequence, in which said template strand consists of RNA from one of positions +1 to +5 up to the 5′ end of the template strand, and consists of DNA from said position up to the 3′ end of the template strand when said 3′ end does not coincide with said position.
Use according to the preceding claim, in which said RNA polymerase is a virus or phage wild-type RNA polymerase.
Use according to the preceding claim, in which said RNA polymerase is chosen from T7, T3 and SP6 RNA polymerase.
Dependencies are inferred from the claim text.