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Method for confirming 3.-terminus sequence of virus RNA (Ribonucleic Acid) molecule

A terminal sequence and virus technology, applied in the direction of DNA preparation, recombinant DNA technology, biochemical equipment and methods, to achieve the effect of saving experiment cost and simplifying experiment cycle

Inactive Publication Date: 2012-08-01
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since a small number of messenger RNA and other types of RNA molecules do not have a polyA structure, the sequence of these RNAs cannot be directly obtained by reverse transcription using Oligo dT primers or modified Oligo dT primers

Method used

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  • Method for confirming 3.-terminus sequence of virus RNA (Ribonucleic Acid) molecule
  • Method for confirming 3.-terminus sequence of virus RNA (Ribonucleic Acid) molecule
  • Method for confirming 3.-terminus sequence of virus RNA (Ribonucleic Acid) molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The method for obtaining the 3' end sequence of rabies virus (Rabies virus) genome is an example, and its steps are as follows:

[0028] 1. Extraction of rabies virus RNA

[0029] Rabies virus (ERA strain) was purchased from China Center for Veterinary Microorganism Culture Collection. After inoculating the hamster kidney cells BHK-21 with the virus, culture them in an incubator at 33-35°C for 5 days. The infected cell culture was collected, and the rabies virus RNA was extracted with the QIAamp Viral RNA Mini Kit kit according to the instructions. The QIAamp Viral RNA Mini Kit kit is a commercially available commodity sold by Qiagen. Rabies virus RNA was dissolved in 40 μL diethyl pyrocarbonate-treated water, 1 μL was taken to test the sample concentration, and the concentration of rabies virus RNA was quantified to 1 μg / μL.

[0030] 2. Terminal transfer reaction of rabies virus RNA

[0031] Add 7.5 μL of rabies virus RNA, 1 μL of deoxyadenosine triphosphate with a ...

Embodiment 2

[0042] Take the method for positioning the 3' end sequence of the influenza virus nucleoprotein (Nucleoprotein, NP) gene as an example, and its steps are as follows:

[0043] 1. Extraction of influenza virus RNA

[0044] Influenza virus (A / Hubei / 1190 / 2010 (H3N2)) was purchased from China Center for Type Culture Collection. After inoculating the chicken embryos with the virus, culture the chicken embryos in a 33-35°C incubator for 2-3 days. The allantoic fluid of chicken embryos was collected, and viral RNA was extracted with the QIAamp Viral RNA Mini Kit kit according to the instructions. The QIAamp Viral RNA Mini Kit kit is a commercially available commodity sold by Qiagen. Viral RNA was dissolved in 40 μL diethylpyrocarbonate-treated water, 1 μL was taken to test the sample concentration, and the concentration of influenza virus RNA was quantified to 1 μg / μL.

[0045] 2. Terminal transfer reaction of influenza virus RNA

[0046]Add 7.5 μg of influenza virus RNA, 1 μL of d...

Embodiment 3

[0057] The method for obtaining the 3' end sequence of the 28S rRNAα subunit of Schistosoma mansoni is an example, and the steps are as follows:

[0058] 1. Extraction of Schistosoma mansoni RNA

[0059] The RNA of Schistosoma mansoni was extracted by one-step method with Tizol reagent, and the quality of the obtained Schistosoma mansoni RNA was detected by formaldehyde denaturing gel electrophoresis. The Tizol reagent was a commercial product sold by Dalian Bao Biological Engineering Co., Ltd. Quantify the concentration of viral RNA to 1 µg / µL.

[0060] 2. Terminal transfer reaction of Schistosoma mansoni RNA

[0061] Add 7.5 μg of Schistosoma mansoni RNA, 1 μL of 10 mmol / L deoxyadenosine triphosphate, diethyl pyrocarbonate-treated water to 12.5 μL, and 10 mmol / L of deoxyadenosine triphosphate and diethyl pyrocarbonate in order to the centrifuge tube The volume ratio of ester-treated water and Schistosoma mansoni RNA is 1:4:7.5, mix well, incubate at 70°C for 15 minutes to ...

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Abstract

A method for confirming a 3.-terminus sequence of a virus RNA (Ribonucleic Acid) molecule comprises the following steps of: extraction of virus RNA, tail transfer reaction of the virus RNA, purification of tailing virus RNA, reverse transcription reaction of the tailing virus RNA, and polymerase chain reaction amplification of a product of the virus reverse transcription reaction. According to the invention, after tailing is performed on the virus RNA by the tail transfer reaction, the 3.-terminus sequence of the virus RNA molecule can be accurately confirmed by the reverse transcription reaction, thus the method is convenient and quick. As an Oligo dT primer or a modified Oligo dT primer is utilized to perform the reverse transcription reaction, the process of designing and compounding a specific primer according to a specific virus RNA molecule is eliminated, so that the period of an experiment is simplified greatly, and the experiment cost is saved. Simultaneously, the method can be applied to confirming other 3.-terminus sequences of the RNA molecule the 3.-terminus of which does not have a PolyA tail structure.

Description

technical field [0001] The invention provides a method for quickly and accurately determining the 3' end sequence of a viral RNA molecule. Background technique [0002] Viruses are non-cellular organisms composed of nucleic acids and proteins, strictly parasitic in cells, and use the host's enzyme system to replicate. Relying on this mechanism, the virus can lead to the occurrence of persistent infection or the transformation of cells to form tumors. At present, the research on viruses mainly focuses on three aspects: on the one hand, to effectively prevent and control the occurrence and prevalence of various viral diseases; on the other hand, to further understand the basic problems of living matter through the understanding of viruses; The three aspects are to use viruses to benefit mankind. But no matter which aspect, the study of viral genetic material is particularly important. [0003] A virus has only one specific type of nucleic acid, either deoxyribonucleic acid ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/70C12Q1/68
Inventor 李治孙燕王立飞孙书洪
Owner SHAANXI NORMAL UNIV
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