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Enhanced magnetic-bead-based nucleic acid extraction method

An extraction method, the technology of magnetic bead method, which is applied in the biological field, can solve the problems that cannot meet the needs of the development of molecular diagnosis business, and achieve the effect of enhancing RNA extraction efficiency, overcoming numerous steps, and efficiently extracting RNA

Active Publication Date: 2011-11-02
薛昱
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the outbreak of SARS in 2003, most of the 1,000 or so tertiary hospitals and 400 prefecture-level disease control centers across the country have been equipped with PCR machines / fluorescent PCR machines, established molecular diagnostic laboratories for pathogenic microorganisms, and carried out extensive research. Molecular diagnostic business including HBV, HCV, HIV, SARS, AIV, H1N1 and other viruses. The huge molecular diagnostic market has cultivated a large number of manufacturers of diagnostic reagents, led by Kehua, Fosun, Da'an, etc. Represented by the company, molecular diagnosis is the main business segment. However, the current virus RNA extraction method represented by silica gel membrane adsorption column method obviously cannot meet the needs of the development of molecular diagnosis business.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1 Experiment purpose

[0046]Use a known amount of λ-DNA (purchased from TAKARA, article number D3010, chain length 48502bp), Salmon Sperm DNA (salmon sperm DNA, purchased from Sigma, article number D1626, chain length about 2000bp) as samples, using the magnetic The bead nucleic acid extraction method carries out DNA extraction, and the DNA extraction efficiency of the present invention is calculated by comparing the amount of DNA obtained by eluting after extraction with the known amount of DNA before extraction.

[0047] 2DNA extraction

[0048] (1) Cleavage and binding: Add 100 μl of a known amount of DNA sample (λ-DNA, or Salmon Sperm DNA) to a solution containing 200 μl lysate, 25 μl proteinase K solution (15-25 mg / ml), 100 μl magnetic beads-isopropanol solution (0.3-0.7mg magnetic beads dissolved in 100μl isopropanol) in a 1.5ml centrifuge tube, mix thoroughly, and place at room temperature for 8min, the free DNA is adsorbed to the surface of the magnetic beads ...

Embodiment 2

[0065] 1 Experiment purpose

[0066] Use hepatitis C positive plasma sample, concentration is respectively A group, 10 times dilution A group, each group concentration extracts 2 parts with the magnetic bead method nucleic acid extraction method that the present invention establishes, and with QIAGEN Viral RNAMini Kit (purchased from Qiagen, product number 52904, silica gel Membrane adsorption column method) extract 1 part, carry out the comparison of two kinds of extraction methods virus RNA extraction efficiency of same sample. The Qiagen Viral RNA Mini Kit based on the silica gel membrane adsorption column method is currently recognized as the gold standard reagent for viral RNA extraction. The operation is according to the instructions, and will not be repeated below.

[0067] 2 Viral RNA extraction

[0068] When the present invention is applied to RNA extraction, all reagents used must be prepared with 1‰ DEPC-treated ultrapure water, and all consumables used must be fre...

Embodiment 3

[0085] 1 Experiment purpose

[0086] Use hepatitis C positive plasma sample, the concentration is B group, 10 times dilution group B respectively, 100 times dilution B group, 1000 times dilution B group, each group concentration extracts 2 parts respectively with the magnetic bead method nucleic acid extraction method that the present invention establishes, simultaneously with MagMAX TM -96 viral RNAIsolation kit (purchased from Ambion, product number AM1836, RNA extraction method by magnetic bead method) was extracted in 2 parts, and the viral RNA extraction efficiency of the two magnetic bead method extraction methods of the same sample was compared.

[0087] Ambion, a subsidiary of Applied Biosystems, is a world-renowned expert in dealing with RNA problems. Its magnetic bead virus RNA extraction reagent MagMAX TM -96 viral RNA Isolation kit is not only launched earlier, but also is a magnetic bead RNA extraction reagent certified by the US FDA for the extraction of avian i...

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Abstract

In the prior art, the magnetic-bead-based nucleic acid extraction method has the following two disadvantages: one disadvantage is that the RNA extraction efficiency is low and the other disadvantage is that the full automatic application is not achieved because the nucleic acid extraction procedures are too fuzzy, the cracking procedure and the bonding procedure are separated and magnetic beads are added to a reaction solution to bond nucleic acids only after the cracking is completed. The invention provides an enhanced magnetic-bead-based nucleic acid extraction method and is characterized in that: the nucleic acid extraction is completed only by the following four steps: cracking and bonding, primary cleaning, secondary cleaning and eluting; in the former three steps, the nucleic acid-adsorbing action of the magnetic beads in the solution is enhanced by adding isopropanol, so as to increase the efficiency of magnetic-bead-based nucleic acid extraction; and a high-efficiency RNA extraction method is provided for the application in RNA extraction. The invention is also characterized in that: the cracking operation and the bonding operation are completed in one step, and during theapplication in automatic nucleic acid extraction, the reaction reagent in each step can be filled in advance into the corresponding reaction chamber of the step, so that the enhanced magnetic-bead-based nucleic acid extraction method is more suitable for full automatic application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an enhanced magnetic bead nucleic acid extraction method, in particular to a method for enhancing nucleic acid extraction efficiency by isopropanol to enhance the adsorption of nucleic acid by magnetic beads in a solution, and the cleavage and combination of nucleic acid extraction The steps are completed in one step to enhance the fully automated application of the magnetic bead nucleic acid extraction method. Background technique [0002] Nucleic acid includes two types of deoxyribonucleic acid (RNA) and ribonucleic acid (DNA). It is known that nucleic acid is the genetic material of all organisms. It mainly exists in the nucleus in cells and in the viral capsid in viruses. Cells are the basic unit of the structure and function of organisms. It is known that all organisms except viruses are composed of cells, but the life activities of viruses must also be reflected in cells. [000...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 薛昱
Owner 薛昱
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