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Kit for extracting free DNA

The technology of a kit and a magnetic separator is applied in the field of high-throughput DNA detection, which can solve the problems of low extraction efficiency, inability to separate large and small fragments in plasma, and inability to separate DNA fragments, and achieves the effects of high recovery rate and simple operation.

Inactive Publication Date: 2015-12-02
GENE CRAB BIOTECH CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional phenol chloroform isoamyl alcohol method for plasma cfDNA extraction is to extract all the DNA in the plasma, which cannot separate the large and small fragments in the plasma, and the extraction efficiency is low, so other means are needed to improve its efficiency
The column extraction method has high extraction efficiency, but it cannot separate DNA fragments of different sizes

Method used

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  • Kit for extracting free DNA
  • Kit for extracting free DNA
  • Kit for extracting free DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 A method of extracting large fragments of free DNA (greater than 500bp) in plasma

[0060] Using different isopropanol volume concentration gradients (2%, 4.5%, 7%, 9.5%, 12%), a one-step method of magnetic bead binding was used to extract large fragments of free DNA.

[0061] Experimental system (100l plasma + 230μl detection reagent), the detection reagent includes proteinase K, magnetic beads, isopropanol and BindingBuffer. Plasma: DNA fragments of known concentration of 127bp and 208bp are mixed into the plasma of healthy people, and the two fragments are used as standards. The selection of fragment size simulates the size of cfDNA in plasma (140-150bp), The final concentration is 0 ng / ml, 7.5 ng / ml, 15 ng / ml, 30 ng / ml, 60 ng / ml, 120 ng / ml. Three parallels were performed for each sample.

[0062] The specific method for extracting large DNA fragments (>500bp) is:

[0063] (1) Add 10 μl proteinase K, 20 μl magnetic beads, (6.6 μl isopropanol and 193.6 μl...

Embodiment 2

[0072] Example 2 A method of extracting small fragments of free DNA (less than 500bp) in plasma

[0073]1. Secondary binding Referring to the method described in Example 1 (i.e. primary binding method), add (20 μl of magnetic beads and 189 μl of isopropanol to the supernatant obtained in step (1) in Example 1, respectively; 20 μl of magnetic beads and 230 μl of isopropanol; 20 μl of magnetic beads and 286 μl of isopropanol; 20 μl of magnetic beads and 350 μl of isopropanol, 20 μl of magnetic beads and 428 μl of isopropanol) Vortex to mix well, isopropanol The volume concentrations of propanol were 36%, 41%, 46%, 51%, and 56%, respectively. Let the centrifuge tube stand at room temperature for 10 min. Place the centrifuge tube on a magnetic separator and let it stand for 2 minutes, then suck off the supernatant with a pipette.

[0074] 2. Washing (1) Add 600 μl WashingBuffer I, blow and beat the magnetic beads 10 times slowly with a pipette to fully resuspend the magnetic bea...

Embodiment 3

[0079] The assembly and application of the kit that embodiment 3 extracts free DNA

[0080] The kit of the present invention contains magnetic beads, proteinase K, isopropanol, BindingBuffer, standard products and other commonly used reagents for washing and eluting magnetic beads.

[0081] According to the record of embodiment 1,2, the working procedure of kit of the present invention is as follows:

[0082] 1. Separation of human peripheral blood plasma or urine.

[0083] 2. One-time binding, magnetic bead washing, drying and elution, and DNA extraction.

[0084] To 100 μl of plasma, add 10 μl of proteinase K, 185 μl of BindingBuffer, 15 μl of isopropanol and 20 μl of magnetic beads, and vortex to mix evenly. Heat the centrifuge tube at 55°C for 10 minutes (during this period, invert and mix once). Place the centrifuge tube on the magnetic separator and let it stand for 2min, pipette the supernatant into another new 1.5ml centrifuge tube, collect the magnetic beads for wa...

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Abstract

The invention provides a kit for extracting free DNA and belongs to the technical field of high-throughput DNA detecting. The kit uses two-step magnetic bead absorption to extract the free DNA in blood plasma or urine, low-concentration isopropanol is used in the first step, and magnetic beads mainly absorb large DNA fragments above 500bp; the concentration of the isopropanol is increased in the second step, the magnetic beads mainly absorb small DNA fragments below 500bp, and the DNA fragments above and below 500bp can be extracted by the two-step method. The invention further provides standard products suitable for the two-step method, and the standard products are DNA fragments of 127bp and 208bp. The kit is applicable to prenatal diagnosis and early-stage tumor screening and promising in application prospect in clinical medicine.

Description

technical field [0001] The invention relates to the technical field of high-throughput DNA detection, in particular to a kit for extracting free DNA in plasma or urine and its application. Background technique [0002] Free DNA (cellfreeDNA, cfDNA) is a kind of extracellular DNA in a cell-free state, mainly composed of single-stranded or double-stranded DNA and a mixture of single-stranded and double-stranded DNA, in the form of DNA-protein complex or free DNA exist. There is also cfDNA in the peripheral blood of healthy people, but the cfDNA content in the peripheral blood of tumor patients is significantly higher than that of healthy people, and there are gene mutations, recombination, and deletions in the peripheral blood DNA of tumor patients. Cell-free DNA in the peripheral blood of cancer patients is mainly released by necrosis or apoptosis of tumor cells, lysis of micrometastases or circulating tumor cells, and vigorously proliferating tumor cells. [0003] The most...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 杜波李明珍代东发胡莹张鑫媛
Owner GENE CRAB BIOTECH CO
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