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A kit and method for separating free nucleic acid in peripheral blood using magnetic microspheres

A technology of magnetic microspheres and free nucleic acid, applied in biochemical equipment and methods, microbiological measurement/testing, DNA preparation, etc., can solve the problems of low degree of automation and high-throughput operation, low extraction efficiency of free nucleic acid, and difficulty in clinical use Large and other problems, to achieve the effect of high-throughput automatic operation, improve extraction and separation efficiency, efficient rinsing and elution

Active Publication Date: 2020-10-09
GENFINE BIOTECH BEIJING CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a kit and method for separating free nucleic acid in peripheral blood using magnetic microspheres to solve the problem of low extraction efficiency, complicated steps, poor repeatability, and high cost of free nucleic acid in the prior art, which is difficult for clinical use. Problems with high difficulty, low degree of automation and high-throughput operations, through the development of non-specific capture probes, the specific binding of Oligo(dT)25 to Poly-A, and the optimization of lysis buffer, binding buffer and washing buffer etc. to achieve fast, efficient and sufficient extraction of free nucleic acid in peripheral blood, with low cost and good repeatability, suitable for routine clinical use, easy to automate and high-throughput operation

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  • A kit and method for separating free nucleic acid in peripheral blood using magnetic microspheres

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Embodiment 1

[0028] Example 1 Separation of Free Nucleic Acids in Plasma Samples Using Magnetic Microspheres

[0029] ① Add 200 μL plasma sample to a nuclease-free centrifuge tube, then add 450 μL lysis buffer to the centrifuge tube and mix well, then add 20 μL proteinase K, blow with a pipette or turn it upside down until fully mixed until uniform, And keep it for 10min to obtain the lysed sample;

[0030] ② Add 100 μL of binding buffer and 30 μL of nucleic acid binding reagent to the lysed sample, the nucleic acid binding reagent contains conjugated Oligo(dT) 25 Aqueous solution of magnetic microspheres and non-specific capture probes with Poly-A, which is coupled with Oligo(dT) 25 The concentration of magnetic microspheres is 0.1-5mg / mL, and Oligo(dT) is coupled to the magnetic microspheres 25 The amount is 50-200nmol / g, and the concentration of non-specific capture probes with Poly-A is 0.5-5nmol / mL. Use a pipette to blow or mix upside down. Let stand at room temperature for 20min. ...

Embodiment 2

[0034] Example 2 Separation of Free Nucleic Acids in Serum Samples Using Magnetic Microspheres

[0035] ① Add 150 μL serum sample to a nuclease-free centrifuge tube, then add 400 μL lysis buffer to the centrifuge tube and mix well, then add 15 μL proteinase K, blow with a pipette or turn it upside down until fully mixed until uniform, And keep it for 6 minutes to get the lysed sample;

[0036] ② Add 100 μL of binding buffer and 35 μL of nucleic acid binding reagent to the lysed sample. The nucleic acid binding reagent is an aqueous solution containing magnetic microspheres coupled with Oligo(dT)25 and non-specific capture probes with Poly-A. Mix evenly by suction or upside down, and let stand at room temperature for 15 minutes. Place the centrifuge tube on the magnetic stand for 1 minute. At this time, the magnetic microspheres are enriched on the magnet and separated from the supernatant. The supernatant is discarded by suction. Obtain the magnetic bead-nucleic acid complex,...

Embodiment 3

[0039] Example 3 Separation of Free Nucleic Acids in Plasma Samples Using Magnetic Microspheres

[0040] ① Add 100 μL plasma sample to a nuclease-free centrifuge tube, then add 400 μL lysis buffer to the centrifuge tube and mix well, then add 15 μL proteinase K, blow with a pipette or turn it upside down until fully mixed until uniform, And keep it for 10min to obtain the lysed sample;

[0041] ② Add 80 μL of binding buffer and 20 μL of nucleic acid binding reagent to the lysed sample, the nucleic acid binding reagent contains conjugated Oligo(dT) 25 The aqueous solution of the magnetic microspheres and the non-specific capture probes with Poly-A was mixed evenly with a pipette or upside down, and left to stand at room temperature for 15 minutes, and the centrifuge tube was placed on a magnetic stand for magnetic adsorption for 1 minute Aspirate and discard the supernatant to obtain the magnetic bead-nucleic acid complex, in which the sequence of the non-specific capture prob...

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Abstract

The invention discloses a kit and a method for separating free nucleic acid in peripheral blood by using magnetic microspheres and belongs to the technical field of biologics. Based on magnetic microspheres coupled with Oligo(dT)25 and a nonspecific capturing probe which comprises Poly-A and a sequence of L(k)2-d(k)5-L(k)2-d(k)5-L(k)2-dT5dA15, nucleic acid in a peripheral blood sample is captured and subjected to magnetic separation, and a splitting buffer liquid, proteinase K, a washing buffer solution and an elution buffer solution of a formula are further optimized, so that the kit and the method for separating free nucleic acid in peripheral blood by using magnetic microspheres have the advantages that rapid splitting and efficient washing and elution are achieved, the free nucleic acid in the peripheral blood sample is efficiently extracted, and sufficient free nucleic acid can be extracted from a small amount of samples; the kit and the method are easy to operate and are safe and environmentally friendly, steps are greatly simplified, the experiment efficiency is improved, good repeatability is achieved, and high-flux automatic operation is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to nucleic acid purification, in particular to a kit and method for separating free nucleic acid in peripheral blood by using magnetic microspheres. Background technique [0002] As early as 1948, Mandel et al. discovered free nucleic acid in peripheral blood. The study found 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, Cancer development and studies of chimerism after bone marrow transplantation. 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, which further provided a basis for the correlation between the two. , cell-free nucleic acid began to be used as a novel biomarker to predict tumorigenesis. In re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2563/143C12Q2563/149C12Q2525/173
Inventor 韩典霖杨亮龚小鹏
Owner GENFINE BIOTECH BEIJING CO LTD
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