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Method and kit for extracting free nucleic acid by using paramagnetic particle method

A free nucleic acid and magnetic bead method, applied in the field of nucleic acid extraction, can solve the problems of small sample volume, low extraction efficiency, complicated operation, etc., and achieve the effects of simple operation, improved extraction purity and low free nucleic acid concentration.

Inactive Publication Date: 2016-02-24
杭州千基生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The cell-free DNA extraction method disclosed in the above-mentioned patent still has the problems of complicated operation, poor repeatability, low extraction efficiency and small amount of extracted samples

Method used

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  • Method and kit for extracting free nucleic acid by using paramagnetic particle method
  • Method and kit for extracting free nucleic acid by using paramagnetic particle method
  • Method and kit for extracting free nucleic acid by using paramagnetic particle method

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Embodiment 1

[0059] In the method for extracting free nucleic acid by the magnetic bead method in this embodiment, leukocyte-free plasma is used as a sample in this embodiment. The magnetic nanoparticles used are superparamagnetic ferric oxide particles or superparamagnetic ferric oxide particles, and the surfaces of the superparamagnetic ferric oxide particles or superparamagnetic ferric oxide particles are respectively modified by hydroxyl or Carboxyl, and coated with silica. The lysing and combining solution includes 0.5M guanidine isothiocyanate, 0.5% tween20, and 10% isopropanol, and the pH value of the lysing and combining solution is 5; the washing solution includes 0.5M sodium perchlorate and 70% prepared by mixing absolute ethanol, and the pH value of the washing liquid is 5. The eluent is a Tris buffer without DNase and RNase enzymes, and the Tris buffer is Tris-HCl with a molar concentration of 10 mM and a pH value of 8.5.

[0060] The specific extraction method is: add 0.3ml ...

Embodiment 2

[0062] In the method for extracting free nucleic acid by the magnetic bead method in this embodiment, leukocyte-free serum is used as a sample in this embodiment. The magnetic nanoparticles used are superparamagnetic ferric oxide particles or superparamagnetic ferric oxide particles, and the surfaces of the superparamagnetic ferric oxide particles or superparamagnetic ferric oxide particles are respectively modified by hydroxyl or Carboxyl, and coated with silica. The lysis binding solution includes 6M guanidine hydrochloride, 5% SDS, 50% ethanol and 0.5M sodium iodide mixed, and the pH value of the lysis binding solution is 7; the washing solution includes 3M guanidine isothiocyanate and 30 % of absolute ethanol is mixed, and the pH value of the washing liquid is 7. The eluent is a Tris buffer without DNase and RNase enzymes, and the Tris buffer is Tris-HCl with a molar concentration of 10 mM and a pH value of 8.5.

[0063]The specific extraction method is: add 7.5ml of lys...

Embodiment 3

[0065] In the method for extracting free nucleic acid by the magnetic bead method in this embodiment, leukocyte-free plasma is used as a sample in this embodiment. The magnetic nanoparticles used are superparamagnetic ferric oxide particles or superparamagnetic ferric oxide particles, and the surfaces of the superparamagnetic ferric oxide particles or superparamagnetic ferric oxide particles are respectively modified by hydroxyl or Carboxyl, and coated with silica. The lysing solution is prepared by mixing 4M guanidine isothiocyanate, 3% tritonX100, 30% isopropanol and 2M sodium perchlorate. The pH value of the lysing solution is 6; the washing solution includes 1.5M guanidine hydrochloride It is prepared by mixing with 50% absolute ethanol, and the pH value of the washing solution is 6. The eluent is a Tris buffer without DNase and RNase enzymes, and the Tris buffer is Tris-HCl with a molar concentration of 10 mM and a pH value of 8.5.

[0066] The specific extraction metho...

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Abstract

The invention relates to a method for extracting a free nucleic acid by using a paramagnetic particle method, which comprises the following steps: adding 300 mu l of a cracked binding solution and 20 mu l of magnetic nanoparticles into 200 mu l of a sample, mixing, then carrying out first magnetic separation on the obtained mixture to remove supernatant obtained after the first magnetic separation is implemented; adding 0.5-1 ml of washing liquid into the obtained object, mixing, carrying out second magnetic separation on the obtained mixture, and removing supernatant obtained after the second magnetic separation is implemented; adding 40-100 mu l of eluent into the obtained object, mixing, standing the obtained mixture for 5-10 min, wherein the standing temperature is 20-65 DEG C; and carrying out third magnetic separation on the obtained object, taking supernatant obtained after the third magnetic separation is implemented, so that the free nucleic acid is obtained. The invention also relates to a kit for extracting the free nucleic acid by using a paramagnetic particle method. The method for extracting the free nucleic acid by using the paramagnetic particle method disclosed by the invention has the advantages that the method is simple in operation, increases the extraction efficiency and the extraction purity, improves the sample quantity, and is applicable to the extraction of nucleic acids with short fragments.

Description

technical field [0001] The invention relates to nucleic acid extraction technology, in particular to a method for extracting free nucleic acid by a magnetic bead method and a kit thereof. Background technique [0002] Since Mandel and Metais first reported free nucleic acid in 1948, the study of free nucleic acid has a history of nearly 70 years, but it has not attracted widespread attention because of whether nucleic acid is genetic material at that time. In 1970, European scholars reported for the first time that free DNA exists in the plasma or plasma of tumor patients, and its content is about 10 times that of healthy people. It is a double-stranded DNA molecule, the length of which is basically less than 500bp, and most of them exist in the form of nucleosomes. A large number of studies have proved that cell-free DNA and tumor genomic DNA have the same genetic variation, such as gene mutation, gene promoter methylation, etc. Therefore, cell-free nucleic acid can be used...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 尹华立郑银娜裘惠良
Owner 杭州千基生物科技有限公司
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