Methods and kit for primer-dependent amplification heterologous viral, eukaryotic and prokaryotic RNA

摘要

An RNA-dependent RNA-polymerase for amplification and/or labeling of RNA which exhibits right-hand-conformation and comprising a defined amino acid sequence, is new. An RNA-dependent RNA-polymerase for amplification and/or labeling of RNA which exhibits right-hand-conformation and comprises the amino acid sequences: X-X-Asp-Tyr Gly-X-Pro-Ser-Gly Tyr-Gly-Asp-Asp X-X-Tyr-Gly-Leu X-X-X-X-X-Phe-Leu-X-Arg-X-X X : any amino acid. Independent claims are included for: (1) a procedure for amplifying RNA by RNA-dependent RNA-polymerase comprising attaching the RNA-dependent RNA-polymerase at the RNA-template strand in the presence of absence of an oligonucleotide primer; transcripting the RNA-template strand in antisense RNA by the RNA-dependent RNA-polymerase; and separating the RNA/antisense-RNA duplex into individual RNA strands; (2) a procedure for primer-dependent amplification of RNA comprising providing at least one primer or non-primer to hybridize an section of the RNA template-strand and attaching the RNA-dependent RNA-polymerase, which extends the primer according to the sequence the RNA-template strand; (3) a procedure for primer-independent amplification of poly-(C)-RNA comprising providing a non-primer to hybridize a section of the RNA-template strand and further providing a guanidine triphosphate (GTP) nucleotide and extending the primer by RNA-dependent RNA-polymerase according to sequence of the RNA-template strand; (4) a procedure for labeling RNA comprising attaching the RNA-dependent RNA-polymerase, to which to be labeled; and attaching at least one nucleotide to the 3'-end of the RNA, which is to labeled; (5) a kit for the amplification of RNA comprising at least the RNA-dependent RNA-polymerase, a suitable reaction buffer, nucleotide triphosphates, optionally RNAse inhibitor, an stop solution and a helicase; (6) a kit for primer-dependent amplification of RNA comprising at least an RNA-dependent RNA-polymerase, a suitable reaction buffer, nucleotide triphosphates, optionally RNAse inhibitor, an stop solution and a primer; (7) a kit for primer-independent amplification of RNA comprising the components excluding the primer; and (8) a kit for labeling of RNA comprising at least an RNA-dependent RNA-polymerase, a suitable reaction buffer, a cytosine-, uridine-, adenine- or guanine-triphosphate of the nucleotide, optionally RNAse inhibitor and an stop solution.