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Rna-dependent rna polymerase, methods and kits for the amplification and/or labelling of rna

An RNA polymerase, kit technology, applied in biochemical equipment and methods, enzymes, transferases, etc.

Inactive Publication Date: 2008-07-23
RIBOXX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Methods for labeling RNA with CTP, UTP and GTP have not been reported yet

Method used

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  • Rna-dependent rna polymerase, methods and kits for the amplification and/or labelling of rna
  • Rna-dependent rna polymerase, methods and kits for the amplification and/or labelling of rna
  • Rna-dependent rna polymerase, methods and kits for the amplification and/or labelling of rna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0211] Preparation method of recombinant norovirus-RdRP.

[0212] The cDNA of Norwalk virus-RdRP was obtained by performing PCR on the clone pUS-NorII (GenBank accession number: AY741811) of Norwalk virus. The cDNA was cloned into the pET-28b(+) vector (Novagen), and the expression vector was sequenced and transformed into E. coliCL21(DE3)pLysS. Cells were incubated at 37°C in Luria-Bertani medium containing kanamycin (50 mg / l). Protein expression was induced by adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM at an optical density of 0.6 (OD600). Then incubate overnight at a temperature of 25°C. Cell extracts obtained from 250 ml of culture were washed once with 4 ml of phosphate buffered saline (PBS) and 1% Triton X 100 (sigma) water. Cells were treated with DNase enzyme (10 U / ml) at 37° C. for 15 minutes, sonicated, and suspended in 40 ml of binding buffer (20 mM Tris / HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole). Centrifuge at 4300 rpm for 40 mi...

Embodiment 2

[0214]Sequence-independent RNA amplification using Norwalk virus-RdRP.

[0215] 0.5-1 μg RNA template, 10 μl reaction buffer (250mM HEPES, pH 8.0, 15mM magnesium acetate, 20mM DTT), 50U RNase inhibitor (RNAsin, Promega), ATP, CTP, GTP, UTP each 0.4mM, Example 1 A reaction mixture (50 μl) consisting of 3 μM norovirus-RdRP prepared in , was reacted at 30° C. for 2 hours. The reaction was terminated by adding 50 μl of stop solution (4M ammonium acetate, 100 mM EDTA). Purification was carried out by phenol-chloroform extraction, or by MEGAclear kit (Ambion) according to the manufacturer's instructions. After staining with ethidium bromide, the transcripts were observed on agarose gel prepared in TBE buffer by UV transmission illumination. Formamide agarose gels can also be used.

Embodiment 3

[0217] Sequence-dependent RNA amplification using the gene-specific RNA primer, Norwalk virus-RdRP.

[0218] 0.5~1μg RNA template, 10μl reaction buffer (250mM HEPES, pH 8.0, 15mM magnesium acetate, 20mM DTT), 50U RNase inhibitor (RNAsin, Promega), ATP, CTP, GTP, UTP each 0.4mM, 0.1~1μM A reaction mixture (50 μl) consisting of gene-specific RNA primers and 3 μM Norwalk virus-RdRP prepared in Example 1 was reacted at 30° C. for 2 hours. The reaction was terminated by adding 50 μl of stop solution (4M ammonium acetate, 100 mM EDTA). Purification was carried out by phenol-chloroform extraction, or by MEGAclear kit (Ambion) according to the manufacturer's instructions. After staining with ethidium bromide, the transcripts were observed on agarose gel prepared in TBE buffer by UV transmission illumination. Formamide agarose gels or urea / polyacrylamide gels can also be used.

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Abstract

The invention relates to an RNA-dependent RNA-polymerase, to methods and kits for marking and / or amplifying in a primary dependent or independent manner a ribonucleic acid (RNA), in particular a viral, eucaryontic, procaryontic and double-stranded ribonucleic acid (RNA) for the biological and medical use thereof. Said RNA-dependent RNA-polymerase comprises a right-hand conformation and the amino acid sequence the following sequence sections: a. XXDYS b. GXPSG c. YGDD d. XXYGL e. XXXXFLXRXX having the following significances: D: aspartate Y: tyrosine S: serine G: glicine P: proline L: leucine F: phenylalanine R: arginine X: any amino acid. The inventive amplifying method is particularly suitable for microarray engineering, siRNA production and for diagnosticating viral infections by detecting viral RNA in patient samples. The inventive marking method is particularly suitable for purifying RNA by means of affinity bonds and for marking RNA used in molecular biology methods for characterising the function and / or structure of a viral, eucaryontic, procaryontic and double-stranded ribonucleic acid (RNA).

Description

technical field [0001] The invention relates to an RNA-dependent RNA polymerase, a method and a kit for marker and / or primer-dependent and independent ribonucleic acid (RNA) amplification. In particular, viral RNA, eukaryotic RNA, prokaryotic RNA and double-stranded ribonucleic acid (RNA) for applications in the fields of biotechnology and medicine. The amplification method involved in the present invention is especially suitable for microarray technology, preparation of siRNA and diagnosis of viral infection by detecting viral RNA in patient samples. The labeling method involved in the present invention is especially suitable for the purification of RNA by affinity ligation, and for the molecular biology of the functional and / or structural characteristics of viral RNA, eukaryotic RNA, prokaryotic RNA and double-stranded ribonucleic acid (RNA) researching. Background technique [0002] RNA is an important component in both eukaryotic and prokaryotic cells. It is involved ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12Q1/68
CPCC12N9/127C12Q1/686C12Q2521/119
Inventor 雅克·罗哈严沃尔弗拉姆·鲁道夫卡特里·耶格尔恩诺·雅各布斯
Owner RIBOXX
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