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Microwave-driven rna polymerization by rna polymerases of caliciviruses

An RNA polymerase and calicivirus technology, which is applied in the field of microwave-driven calicivirus RNA polymerase RNA polymerization, and can solve the problem of not providing

Inactive Publication Date: 2013-05-22
RIBOXX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no data are presented that do show that DNA polymerases perform successful DNA polymerization reactions under microwave irradiation

Method used

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  • Microwave-driven rna polymerization by rna polymerases of caliciviruses
  • Microwave-driven rna polymerization by rna polymerases of caliciviruses
  • Microwave-driven rna polymerization by rna polymerases of caliciviruses

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0074] Example 1: Microwave-driven primer-independent de novo priming of RNA synthesis and double-stranded RNA generation by sapovirus RNA polymerase

[0075] Sapovirus RNA polymerase (SEQ ID NO:11) and RNA template (5'-AUACCUAGAAUCUGACCAACCCCC-3'; SEQ ID NO:15) were irradiated with microwaves at 800W for 60s in a conventional microwave oven. The sapovirus RNA polymerase uses single-stranded RNA as a template to generate double-stranded RNA (Figure 1, lane 2). The resulting product is incubated with S1 nuclease. The product was not digested after incubation with S1 nuclease (Figure 1, lane 3), indicating the double-stranded nature of the product. All reactions were performed in a total volume of 25 μl. The RNA polymerase reaction mix contains 1 μg template, 7.5 μM RNA polymerase, each of 0.4 mM ATP, CTP, UTP, and 2 mM GTP, 5 μl reaction buffer (HEPES 250 mM, MnCl 2 25mM, DTT 5mM, pH7.6), and RNAse-DNAse-free water to make up to a total volume of 25μl. For S1 nuclease dige...

example 2

[0076] Example 2: Microwave Power and Irradiation Time Required for Microwave-Driven RNA Synthesis by Calicivirus RNA Polymerase

[0077] Sapovirus RNA polymerase (SEQ ID NO:11) was incubated with RNA template (5'-AUACCUAGAAUCUGACCAACCCCC-3'; SEQ ID NO:15). The reaction was irradiated in a conventional microwave oven at 80W, 160W, 240W, 320W, 400W, 480W, 560W, 640W, 720W and 800W for 60 s in a total volume of 25 μl. In another experiment, the reactant was irradiated for 60s, 30s, 15s, or 5s in the same microwave oven at 80W (as shown in Figure 2B, lanes 1-4), or irradiated at 800W for 60s, 30s, 15s, or 5s ( Figure 2B, lanes 5-8). Products of the expected size were produced in all reactions (Fig. 2A, 2B).

[0078] The reaction mix contains 1 μg template, 7.5 μM RNA polymerase, each of 0.4 mM ATP, CTP, UTP, and 2 mM GTP, 5 μl reaction buffer (HEPES 250 mM, MnCl 2 25mM, DTT 5mM, pH 7.6), and RNAse-DNAse-free water to make up to a total volume of 25μl. The product was separat...

example 3

[0079] Example 3: Calicivirus RNA polymerase de novo primes RNA synthesis in a primer-independent manner using DNA as a template under microwave irradiation, and incorporates 2'-fluoro-GMP into double-stranded DNA / RNA products

[0080] Sapovirus RNA polymerase (SEQ ID NO:11) was incubated with ssDNA template (5'-ATACCTAGAATCTGACCAACCCCC-3'; SEQ ID NO:16) or with the same sequence but with (rC) at the 3' end 5 DNA template for the motif (5'-ATACCTAGAATCTGACCAArCrCrCrCrC-3'; SEQ ID NO: 17). As a control, sapovirus RNA polymerase was incubated with single-stranded RNA (5'-AUACCUAGAAUCUGACCAACCCCC-3'; SEQ ID NO: 15) having the same sequence as the aforementioned single-stranded DNA. All reactions were irradiated in a conventional microwave oven at 800W for 60 s in a total volume of 25 μl. The reaction mix contained 1 μg template, 7.5 μM RNA polymerase, 0.4 mM each of ATP, CTP, UTP, and GTP (using ssRNA template as a control reaction; Figure 3, lane 3) or 2'-fluoro-GTP (reaction ...

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Abstract

The present invention relates to a method for polymerising a complementary RNA strand on a single-stranded polynucleotide template comprising the step of irradiating a composition containing said template and an RNA polymerase of a virus of the Caliciviridae family under RNA polymerisation conditions in the presence or absence of a primer hybridised to the template, with an effective amount of microwave energy. Further subject matter of the invention relates to a method for transferring one or more ribonucleotides to the 3' end of a single-stranded polynucleotide template comprising the step of irradiating a composition containing an RNA polymerase of a virus of the Caliciviridae family in the presence of rATP or rGTP or rUTP or rCTP or a modified or labelled analogue thereof with an effective amount of microwave energy.

Description

technical field [0001] The present invention relates to a method for synthesizing a complementary RNA chain using a single-stranded polynucleotide as a template. The method comprises the following steps: using an effective amount of microwave energy to inactivate RNA polymerase containing the template and a Caliciviridae family virus The mixture is irradiated under RNA polymerization conditions and in the presence or absence of primers that hybridize to the template. Another subject of the invention relates to a method for transferring one or more nucleotides to the 3' end of a single-stranded polynucleotide template, comprising the steps of: applying an effective amount of microwave energy to a The mixture of RNA polymerases of the family Virus is irradiated in the presence of rATP or rGTP or rUTP or rCTP or modified or labeled analogues thereof. Background technique [0002] The RNA-dependent RNA polymerase (hereinafter referred to as "RNA polymerase") of viruses of the C...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6853C12P19/34C12Q2525/101C12Q2523/313C12Q2521/119
Inventor 贾克斯·洛哈耶姆
Owner RIBOXX
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