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Nucleic acid extraction composition and application thereof, reagents and kits containing the nucleic acid extraction composition

A composition and reagent technology, applied in the field of nucleic acid extraction composition, can solve problems such as cumbersome operation, unsuitable use, and harmfulness, and achieve the effects of simple operation, improved timeliness, and solving hazards

Active Publication Date: 2020-08-04
CAPITALBIO CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Trizol method separates proteins and nucleic acids by using the difference in the distribution positions of DNA, RNA, and proteins in the organic layer and the aqueous layer. In the process of obtaining nucleic acids, phenol, chloroform, isoamyl alcohol, isopropanol, and ethanol are used, which are harmful to the human body. The volatile organic solvents present great health hazards to operators, and the Trizol method is cumbersome to extract nucleic acids, and the reproducibility of different sample extraction results is not good. It takes at least 1.5 hours from sample lysis to nucleic acid acquisition; , cumbersome operation, time-consuming, incompatible with the chip and other shortcomings, it is not suitable for clinical testing
The conventional centrifugal adsorption column and magnetic bead method also need to use ethanol or isopropanol for binding when extracting nucleic acid, and ethanol needs to be added when cleaning; this method of adding alcohol makes the operation of nucleic acid extraction reagents relatively complicated and time-consuming. Long, and the reagents added with alcohol cannot be stored for a long time, and the subsequent reaction will continue if the ethanol is not removed cleanly, and the extraction and purification reagents containing ethanol are not suitable for use on microfluidic chips

Method used

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  • Nucleic acid extraction composition and application thereof, reagents and kits containing the nucleic acid extraction composition
  • Nucleic acid extraction composition and application thereof, reagents and kits containing the nucleic acid extraction composition
  • Nucleic acid extraction composition and application thereof, reagents and kits containing the nucleic acid extraction composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Nucleic acid extraction reagents include:

[0050] Lysis solution: the concentration of guanidine isothiocyanate is 4M, the concentration of TritonX-100 is 20% (v / v), the solution pH=6.0

[0051] Binding solution: Glycogen concentration is 0.5% (m / v), MOPS concentration is 20mM, spermine concentration is 0.5% (m / v), TCEP concentration is 6mM, pH=9.5.

[0052] Cleaning solution: the concentration of guanidine hydrochloride is 0.1M, the concentration of methyl acetate is 0.15M, the concentration of glycogen is 0.05% (m / v), the concentration of MOPS is 2mM, the concentration of spermine is 0.2% (m / v), pH=6.0.

[0053] Enhancer: the concentration of proteinase K is 20mg / ml, the concentration of trehalose is 6% (m / v), and the concentration of calcium chloride is 5mM.

[0054] Eluent: 1×TE pH=10.0.

[0055] Extraction effect: Dilute the EV71 pseudovirus constructed by Xiamen Zhishan with saliva (the sample comes from Boao Biohealth employees), so that the concentration of t...

Embodiment 2

[0065] Lysis solution: the concentration of guanidine isothiocyanate is 6M, the concentration of TritonX-100 is 20% (v / v), the solution pH=6.0

[0066] Binding solution: glycogen concentration is 0.1% (m / v), MOPS concentration is 20mM, spermine concentration is 0.1% (m / v), TCEP concentration is 10mM, pH=9.6.

[0067] Cleaning solution: the concentration of guanidine hydrochloride is 0.05M, the concentration of methyl acetate is 0.05M, the concentration of glycogen is 0.01% (m / v), the concentration of MOPS is 1mM, the concentration of spermine is 0.01% (m / v), pH=6.0.

[0068] Enhancer: the concentration of proteinase K is 20mg / ml, the concentration of trehalose is 6% (m / v), and the concentration of calcium chloride is 5mM.

[0069] Eluent: 1×TE pH=10.0.

[0070] Extraction effect: Dilute the EV71 pseudovirus constructed by Xiamen Zhishan with saliva (the sample comes from Boao Biohealth employees), so that the concentration of the pseudovirus in the saliva is 1000 copies / µL, 1...

Embodiment 3

[0080] Lysis solution: the concentration of guanidine isothiocyanate is 6M, the concentration of TritonX-100 is 5% (v / v), and the solution pH=6.0.

[0081] Binding solution: glycogen concentration is 1% (m / v), MOPS concentration is 20mM, spermine concentration is 2% (m / v), TCEP concentration is 10mM, pH=9.6.

[0082] Cleaning solution: the concentration of guanidine hydrochloride is 0.6M, the concentration of methyl acetate is 0.5M, the concentration of glycogen is 0.1% (m / v), the concentration of MOPS is 2mM, the concentration of spermine is 0.3% (m / v), pH=5.0.

[0083] Enhancer: the concentration of proteinase K is 20mg / ml, the concentration of trehalose is 6% (m / v), and the concentration of calcium chloride is 5mM.

[0084] Eluent: 1×TE pH=10.0.

[0085] Extraction effect: Dilute the EV71 pseudovirus constructed by Xiamen Zhishan with saliva (the sample comes from Boao Biohealth employees), so that the concentration of the pseudovirus in the saliva is 1000 copies / µL, 100 c...

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Abstract

The invention relates to the field of biotechnology, in particular to a nucleic acid extraction composition and its application, reagents and kits containing the nucleic acid extraction composition. The invention provides a set of nucleic acid extraction and purification reagents that do not contain volatile organic solvents, which solves the harmfulness of volatile organic solvents to the human body, makes the operation extremely simple, and greatly improves the timeliness of nucleic acid extraction and purification. Nucleic acid can be obtained within 10 minutes, and the obtained nucleic acid can be used in biological reactions such as PCR, NASBA, LAMP, RPA, etc., and the reagent of the present invention can be obtained from blood, throat swab preservation solution, saliva, urine, sputum, feces, etc. Obtain nucleic acids of cells, bacteria, fungi, DNA viruses, and RNA viruses from a variety of complex samples, which is very suitable for clinical and scientific research applications.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid extraction composition and its application, reagents and kits containing the nucleic acid extraction composition. Background technique [0002] Nucleic acid extraction technology is the first step to complete all molecular reactions, and the quality of this step directly affects the success of subsequent biological reactions. Nucleic acid extraction and purification technology is a basic technology in molecular biology experiments. Nucleic acid extraction is inseparable from the initial target gene extraction to PCR product purification or "gel recovery", as well as the extraction of plasmids in gene cloning. purification. Nucleic acid separation and purification technology originated in the 1950s. In the 1970s and 1980s, the traditional nucleic acid precipitation, dissolution, separation and purification method was widely used and promoted. [0003] Commonly used nu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1006C12Q1/6806C12Q2527/125C12N15/1003
Inventor 陈翔王磊李素郑隆堂王亚南文飞辛娟张闻天程京
Owner CAPITALBIO CORP