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Efficient plasma cell dissociation DNA extraction method based on paramagnetic particle method

An extraction method, the technology of magnetic bead method, applied in the biological field, can solve the problems of low extraction efficiency, easy loss of DNA fragments, uneven batch operation effect, etc., and achieve the effect of promoting adsorption and improving extraction efficiency

Inactive Publication Date: 2016-06-15
苏州摩根基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are two main methods for extracting cell-free DNA from plasma: spin column method and magnetic bead method, but in practical applications, there are problems of low extraction efficiency, easy loss of some DNA fragments, and uneven batch operation results. This has a detrimental effect on research applications of plasma cell-free DNA

Method used

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  • Efficient plasma cell dissociation DNA extraction method based on paramagnetic particle method
  • Efficient plasma cell dissociation DNA extraction method based on paramagnetic particle method
  • Efficient plasma cell dissociation DNA extraction method based on paramagnetic particle method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A method for extracting high-efficiency plasma cell free DNA based on the magnetic bead method, characterized in that the specific steps are as follows:

[0025] (1) Pretreatment

[0026] Set up three parallel experiments. Take 200 μl of fresh plasma samples from healthy people and place them in a centrifuge tube, add 20 μl of pretreatment solution P, and store in a water bath at 50°C for 0.5h;

[0027] (2) Adsorption

[0028] Add 800 μl of binding solution B to the pretreated sample; vortex for 30 seconds; react in a 50°C water bath for 5 minutes; take out the centrifuge tube from the water bath and vortex for 15 seconds; use a rotary mixer to react for 10 minutes at room temperature; Finally, centrifuge at 4000g for 30s, put the centrifuge tube on the magnetic stand, and let it stand for 2min to separate the magnetic beads. After the timing is over, remove the supernatant with a pipette and keep the magnetic beads;

[0029] (3) washing

[0030] The first step is s...

Embodiment 2

[0043] When configuring binding solution B in step (1), an equivalent amount of isopropanol was used instead of n-hexane, and the other steps and dosage were the same as in Example 1. Three groups of substitution comparison tests were carried out, and the DNA yield decreased by about 22.3%. The results are shown in Table 2. Show.

[0044] Table 2 Extraction results of plasma cell free DNA

[0045]

Embodiment 3

[0047] In step (2), the DNA adsorption process was changed to vortex oscillation for 30 s; reacted at room temperature for 15 min, and the other steps and dosage were the same as in Example 1. Three sets of alternative comparison tests were carried out, and the DNA yield decreased by about 16.5%. The results are shown in Table 3 shown.

[0048] Table 3 Extraction results of plasma cell free DNA

[0049]

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PUM

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Abstract

The invention discloses an efficient plasma cell dissociation DNA extraction method based on a paramagnetic particle method. The efficient plasma cell dissociation DNA extraction method comprises the steps that firstly, pretreatment liquid P is prepared to preprocess a plasma sample; secondly, combination liquid B is prepared, and DNA in the sample is adsorbed through hydroxyl magnetic beads in a specific mode under the two-step temperature control condition; thirdly, washing liquid W1 and washing liquid W2 are prepared to wash the magnetic beads to which DNA is adsorbed; fourthly, efficient eluting of the sample is achieved under the auxiliary condition of physical reinforcement effect. By means of the efficient plasma cell dissociation DNA extraction method, high-quality cell dissociation DNA can be efficiently and quickly obtained, and the requirements of molecular biological experiments of PCR amplification, quantitative and qualitative analysis, sequencing and the like can be met.

Description

technical field [0001] The invention relates to a high-efficiency plasma cell free DNA extraction method based on a magnetic bead method, belonging to the field of biotechnology. Background technique [0002] Plasma is the liquid component of blood except blood cells and platelets, accounting for about 50-60% of the whole blood volume. Blood is collected through anticoagulant blood collection tubes, and the upper layer of light yellow liquid collected after centrifugation is plasma. Studies have shown that in addition to chemical components such as water, plasma proteins, amino acids, sugars and inorganic salts, there are also deoxyribonucleic acid molecules free from cells in plasma, which are called plasma cell free DNA. In recent years, the application of plasma cell-free DNA in medical diagnosis has been continuously expanded, and it is widely used in non-invasive prenatal screening, tumor liquid biopsy, and individualized drug guidance for tumors and other fields. [...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 王剑青
Owner 苏州摩根基因科技有限公司
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