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Method for separating free nucleic acid from blood serum or blood plasma sample by using magnetic bead

A technology of free nucleic acid and magnetic beads, which is applied in the field of nucleic acid purification, can solve the problems of poor instrument universality, cumbersome steps, and increased costs, and achieve the effect of saving time and simplifying steps

Inactive Publication Date: 2014-07-30
TIANGEN BIOTECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the commercialized magnetic bead method free nucleic acid extraction kit is complicated to operate. It is necessary to digest the sample with proteinase K and lysis buffer at 56°C, then cool it to room temperature, add absolute ethanol and mix well, and then add it after 5 minutes at room temperature. The magnetic bead suspension is mixed, after a series of rinsing steps, it is left to dry at room temperature for 10-15 minutes
The cumbersomeness of the steps leads to a low degree of automation, poor instrument universality, and the reproducibility of nucleic acid extraction cannot be guaranteed
In addition, many magnetic bead method free nucleic acid extraction kits have low extraction efficiency for free nucleic acid from serum or plasma samples, which leads to the need for more initial sample volume or additional carrier RNA or glycogen in the extraction process to meet subsequent detection The requirements not only increase the cost, but also increase the difficulty of clinical application

Method used

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  • Method for separating free nucleic acid from blood serum or blood plasma sample by using magnetic bead
  • Method for separating free nucleic acid from blood serum or blood plasma sample by using magnetic bead
  • Method for separating free nucleic acid from blood serum or blood plasma sample by using magnetic bead

Examples

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Embodiment 1

[0059] Example 1: The embodiment of extracting free nucleic acid from 200 microliters of serum samples

[0060] 1. Add 200 μl serum / plasma sample and 300 μl lysis buffer to a 1.5 ml nuclease-free centrifuge tube;

[0061] 2. Add 20 microliters of proteinase K and 15 microliters of magnetic bead suspension (shake well before use) to the above mixture, mix well, blow with a pipette or mix upside down for 10 minutes at room temperature;

[0062] 3. Place the centrifuge tube in step 2 on the magnetic stand, and magnetically adsorb for 1 minute, so that the magnetic beads are completely adsorbed on the wall of the centrifuge tube, and discard the supernatant;

[0063] 4. Remove the centrifuge tube in step 3 from the magnetic stand, add 500-600 microliters of the first washing buffer, and mix with a pipette or vortex. Place the centrifuge tube on the magnetic stand, magnetically adsorb for 1 minute, and discard the supernatant;

[0064] 5. Remove the centrifuge tube in step 4 from...

Embodiment 2

[0091] Example 2: The implementation of extracting free nucleic acid from 200 microliters of plasma samples:

[0092] 1. Add 200 microliters of plasma sample and 300 microliters of lysis buffer to a 5 milliliter nuclease-free centrifuge tube;

[0093] 2. Add 20 microliters of proteinase K and 15 microliters of magnetic bead suspension (shake well before use) to the above mixture, mix well, blow with a pipette or mix upside down for 10 minutes at room temperature;

[0094] 3. Place the centrifuge tube in step 2 on the magnetic stand, and magnetically adsorb for 1 minute, so that the magnetic beads are completely adsorbed on the wall of the centrifuge tube, and discard the supernatant.

[0095] 4. Remove the centrifuge tube in step 3 from the magnetic stand, add 500-600 microliters of the first washing buffer, and mix with a pipette or vortex. Place the centrifuge tube on the magnetic stand, magnetically adsorb for 1 minute, and discard the supernatant;

[0096] 5. Remove the ...

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Abstract

The invention relates to a method for separating free nucleic acid from a blood serum or blood plasma sample by using a magnetic bead, and belongs to the field of nucleic acid purification. The method comprises the following steps: firstly adding the blood serum / blood plasma sample, a lysis buffer, protease K and magnetic bead suspension liquid into a nuclease-free centrifugal tube, fully and uniformly mixing, then performing pyrolysis, adsorbing nucleic acid on the surface of the magnetic bead, performing purification under the effect of an external magnetic field, removing impurities such as protein, polysaccharide, salt and the like through a rinsing step, and finally eluting by adding nuclease-free water to obtain a free nucleic acid solution. The method disclosed by the invention has the characteristics of simplicity, high speed, stability, high efficiency and cheap price; the purified free nucleic acid is complete and high in purity, and can be directly used for subsequent experiments.

Description

technical field [0001] The invention relates to a method for separating free nucleic acid from serum or plasma samples by using magnetic beads, belonging to the field of nucleic acid purification. Background technique [0002] As early as 1948, Mandel et al. discovered free nucleic acid in peripheral blood, but at that time it did not attract people's attention because it was not yet determined whether nucleic acid was genetic material. With the deepening of research, scientists realized that free nucleic acid can not only be used in the diagnosis and prevention of fetal sex, RHD blood type, and Y-chromosome-linked genetic diseases, but also in the detection of tumors, cancers, and chimerism after bone marrow transplantation. state research. In 1989, Stroun et al. showed that some genes in free nucleic acid had similar characteristics to tumor tissue genes; a few years later, Vasioukhin found mutated N-ras in plasma free nucleic acid, further providing a basis for the corre...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 韩典霖俞萍李晓晨孙克非
Owner TIANGEN BIOTECH BEIJING
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