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Method for fast amplifying doulbe-chain RNA target sequence

A target sequence, rapid technology, applied in the field of double-stranded RNA target sequence amplification, to achieve the effect of short time, less reagents and simple operation

Inactive Publication Date: 2007-04-04
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on direct PCR amplification of a specific target sequence using double-stranded RNA as a template using Taq enzyme

Method used

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  • Method for fast amplifying doulbe-chain RNA target sequence
  • Method for fast amplifying doulbe-chain RNA target sequence
  • Method for fast amplifying doulbe-chain RNA target sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Cucumber mosaic virus (CMV) and its satellite RNA sequence

[0036] 1. Preparation of dsRNA

[0037] (1) Take by weighing 5g of the veined cucumber fresh leaf tissue, fully grind to powder in liquid nitrogen;

[0038] (2) Add 10ml 2 times STE (a mixture of ethylenediaminetetraacetic acid, tris-hydroxy-methyl-aminomethane and sodium chloride, pH8.0), 300μl β-mercaptoethanol, 6ml saturated phenol, 4ml chloroform, 1.4ml of 10% SDS (sodium dodecylsulfonate), fully homogenized for 20 minutes;

[0039] (3) Centrifuge at 15 000 g for 15 min at 4°C, and collect the supernatant;

[0040] (4) The supernatant was adjusted to RNA mixture containing 17% ethanol with absolute ethanol, and mixed at room temperature;

[0041] (5) Weigh 3g of cellulose (CF-11, product of Sigma Company), equilibrate with 1 times STE containing 17% ethanol, and then pack into a column, using a 50ml disposable plastic syringe or equivalent material as a chromatographic column;

[0042] (6...

Embodiment 2

[0060] Example 2: Cucumber mosaic virus satellite RNA sequence

[0061] 1. Preparation of dsRNA

[0062] (1) Take by weighing 5 g of deveined fresh leaf tissue, fully grind to powder in liquid nitrogen;

[0063] (2) Add 10ml 2X STE, 300μl β-mercaptoethanol, 6ml saturated phenol, 4ml chloroform, 1.4ml 10% SDS, and fully homogenize for 20min;

[0064] (3) Centrifuge at 15 000 g for 15 min at 4°C, and collect the supernatant;

[0065] (4) The supernatant was adjusted to RNA mixture containing 17% ethanol with absolute ethanol, and mixed at room temperature;

[0066] (5) Weigh 3g of cellulose (CF-11, product of Sigma Company), equilibrate with 1 times STE containing 17% ethanol, and then pack into a column, using a 50ml disposable plastic syringe or equivalent material as a chromatographic column;

[0067] (6) After loading the RNA mixture, wash the column with 90 ml of 1 times STE containing 17% ethanol;

[0068] (7) Elute with 1X STE without ethanol, discard the first 5ml, c...

Embodiment 3

[0085] Example 3: Cucumber mosaic virus and its satellite RNA sequences.

[0086] 1. Preparation of dsRNA

[0087] (1) Take by weighing 5 g of deveined fresh leaf tissue, fully grind to powder in liquid nitrogen;

[0088] (2) Add 10ml 2X STE, 300μl β-mercaptoethanol, 6ml saturated phenol, 4ml chloroform, 1.4ml 10% SDS, and fully homogenize for 20min;

[0089] (3) Centrifuge at 15 000 g for 15 min at 4°C, and collect the supernatant;

[0090] (4) The supernatant was adjusted to RNA mixture containing 17% ethanol with absolute ethanol, and mixed at room temperature;

[0091] (5) Weigh 3g of cellulose (CF-11, product of Sigma Company), equilibrate with 1 times STE containing 17% ethanol, and then pack into a column, using a 50ml disposable plastic syringe or equivalent material as a chromatographic column;

[0092] (6) After loading the RNA mixture, wash the column with 90 ml of 1 times STE containing 17% ethanol;

[0093] (7) Elute with 1X STE without ethanol, discard the fi...

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PUM

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Abstract

A method for expanding double-chain RNA target sequence fast is carried out by preparing dsRNA, tapping while recovering by agarose electrophoresis, separating and purifying for dsRNA, using Primer 5.0 software to obtain double-chain RNA related primer by GenBank virus gene group related sequence, taking recovered dsRNA as template, adding into double-chain RNA related primer, and PCR expanding under action of Taq enzyme to obtain the final product. It's cheap and simple, has better dsRNA stability and less reagent and saves time and powder.

Description

technical field [0001] The invention relates to double-stranded RNA target sequence amplification, especially direct amplification using dsRNA as a template. Background technique [0002] Viruses are the smallest pathogenic microorganisms parasitic in cells with infective activity composed of nucleic acid and protein shell. According to the type of nucleic acid, it can be divided into DNA virus and RNA virus. RNA viruses are further divided into double-stranded RNA (dsRNA) viruses and single-stranded RNA (ssRNA) viruses. In the process of nucleic acid replication of single-stranded RNA virus, its complementary strand is used as a template to replicate nucleic acid under the action of RNA polymerase (RNA-dependent RNA Polymerase, RDRP). Therefore, during the infection process of RNA viruses, most of them will have double-stranded RNA (double-stranded RNA, dsRNA) replication intermediates, or a way of existence of genetic material. The analysis method of dsRNA has been wide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/68
Inventor 陈集双唐香山竺锡武陈绍宁刘伟侠廖乾生冯俊丽
Owner ZHEJIANG SCI-TECH UNIV
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