Method for isolating highly pure nucleic acid with magnetic particles

A magnetic particle and nucleic acid technology, applied in the preparation of test samples, DNA preparation, recombinant DNA technology, etc.

Active Publication Date: 2019-08-02
QIAGEN GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, this method is not suitable for quantitative nucleic acid isolation

Method used

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  • Method for isolating highly pure nucleic acid with magnetic particles
  • Method for isolating highly pure nucleic acid with magnetic particles
  • Method for isolating highly pure nucleic acid with magnetic particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 and 2

[0105] Examples 1 and 2: Purification of DNA:

[0106] DNA from bacterial cells was isolated according to the method of the invention (Example 2) and the method of the prior art (Example 1). Conditions and working solutions are shown in Table 1 below. use the trademark for (Qiagen, Hilden, Germany; Cat. No. 51304) Commercial kits were used for lysis of E. coli or whole blood cells according to standard protocols. use The kit uses a magnetic bead extraction platform (trademark Gilson Gilson Inc., Middleton, US / SalusDiscovery) was used to purify DNA using magnetic particles. In the standard method of Example 1, DNA is bound to magnetic silica particles from a buffer containing a high concentration of chaotropic salts to establish binding conditions. Applying a magnetic field separates the magnetic particles from the sample solution. In particular, the magnetic particles are drawn out of solution and the sample solution is subsequently removed. In lysis step 1 (table)...

Embodiment 3

[0114] Example 3: Purity of genomic DNA from E. coli

[0115] The impurities in the Escherichia coli genomic DNA prepared in Examples 1 and 2 above were determined by spectrophotometry. To wash away contaminants from the magnetic particles, the magnetic particles were eluted by placing them in a magnetic stirrer for three minutes. The eluate was analyzed for impurities with a spectrophotometer at 220nm-350nm, where most salt residues were determined. The results are shown in figure 1 . The dotted line shows the values ​​obtained for the detection of DNA isolated according to the water-rinsing protocol of Example 1 with the two probes (comparison). The solid line shows the values ​​obtained for DNA isolated according to the water-rinse protocol of Example 2 detected with both probes. High OD values ​​between 220-245nm usually indicate salt and other contaminants. The results show that the inventive method of washing the DNA-bound magnetic particles with distilled water ...

Embodiment 4

[0116] Example 4: Purity of DNA from blood

[0117] DNA from human whole blood was prepared according to the protocols of Inventive Example 2 and a modified Comparative Example 1. In the method of Example 1 above, the third wash step of the water-rinse step 5a (see table) was replaced with PE buffer. During the first and second PE buffer wash steps 3, 4 and the third PE buffer wash step, the magnetic particles fell into solution three times. The purity of the isolated DNA products was compared. The goal was to determine whether the additional washing step in the comparative method (3 drops into ethanol buffer PE) could remove impurities more efficiently than the inventive method in which the particles were only dropped into water once.

[0118] The results are shown in figure 2 . Dashed lines indicate DNA isolated by a comparative triple wash method. The solid line indicates the DNA of Example 2 isolated by the water-drop method. The results show that the method of th...

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PUM

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Abstract

Subject of the invention is a method for isolating nucleic acid from a sample solution, wherein the nucleic acid is separated from the sample solution by adsorption to magnetic particles, followed byseparation of the magnetic particles with the nucleic acid adsorbed thereto from the sample solution by application of a magnetic field, followed by at least one washing step (w), comprising (w1) suspending the magnetic particles with the nucleic acid adsorbed thereto in a washing solution, which is an aqueous solution, which has a total salt concentration below 100 mM and does not comprise an organic solvent, (w2) incubating the magnetic particles with the nucleic acid adsorbed thereto in the washing solution, and (w3) separating the magnetic particles with the nucleic acid adsorbed thereto from the washing solution. The invention also relates to use of an aqueous solution, which has a total salt concentration below 100 mM and does not comprise an organic solvent, as a washing solution for washing magnetic particles with nucleic acid adsorbed thereto.

Description

[0001] The present invention relates to a method for isolating nucleic acid from a sample solution, wherein the nucleic acid is separated from the sample solution by adsorption to magnetic particles, and then the magnetic particles having the nucleic acid adsorbed thereto are separated from the sample solution by applying a magnetic field. A subject of the invention may also be the use of wash buffers in these methods. Background technique [0002] Obtaining DNA or RNA that contains sufficiently few contaminants for molecular biology applications is rather difficult due to the complex systems in which DNA or RNA is typically present. These systems, such as biological samples such as tissues, cells from body fluids such as blood, cultured cells or artificial systems such as agarose gels or PCR reaction samples, often contain considerable amounts of contaminants that should be sensed prior to use in molecular biological processes. The DNA or RNA of interest must be isolated from...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013G01N1/34G01N1/40
Inventor J·斯特洛埃德C·霍克斯坦
Owner QIAGEN GMBH
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