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Magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and application method thereof

A kit and magnetic bead method, applied in the field of molecular biology, can solve problems such as failure to remove genomic interference, inability to remove DNA, and complicated operations, achieving extraction efficiency and purity assurance, saving time and cost, and high extraction efficiency. Effect

Inactive Publication Date: 2016-06-22
SUZHOU ENRICHING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, most of the extraction of cfDNA by magnetic beads can only be carried out in plasma or serum. There are still many difficulties in purifying cfDNA from whole blood. The plasma needs to be centrifuged at 3-6°C and 1600g for 8-12min, and then the supernatant can be centrifuged again at 3-6°C and 16000g for 8-12min to obtain the plasma. The operation is very complicated; however, the serum obtained according to the above method Residual genomic DNA and some larger fragments of DNA cannot be completely removed in
Moreover, there will be 240-800bp DNA fragments mixed in it, and the interference of the genome cannot be removed
Although there are existing technologies that can provide a method for extracting DNA fragments less than 500bp from serum, it still requires complex multi-stage processing of blood to obtain plasma or serum, and the process is complicated

Method used

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  • Magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and application method thereof
  • Magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] A kit for extracting cell-free DNA from blood by magnetic bead method includes separately packaged:

[0043] DNA fixation binding solution: 0.05mol / L urea, Tris base concentration of 100mmol / L, sodium sulfite concentration of 1%, 1mol / L guanidine hydrochloride, 3mol / L guanidine thiocyanate, 2mol / L sodium perchlorate, 100mmol / L Disodium edetate, 0.01% TritonX-100, 1% NP-40, 0.5% sodium lauryl sulfate, 0.1 mol / L potassium acetate and 0.1 mol / L sodium acetate.

[0044] The first magnetic bead suspension: the magnetic bead suspension with a magnetic bead concentration of 40mg / mL, the inner core of the magnetic bead is ferric oxide (Fe 3 o 4 ), the surface is coated with a silicon hydroxyl layer. Specific preparation method: Weigh 4g of magnetic beads, add to 100mL of deionized water, and disperse evenly by ultrasonic.

[0045] Cleaning solution: 60% isopropanol, 50mmol / L disodium edetate, 0.2mol / L guanidine hydrochloride and 0.5mol / L guanidine isothiocyanate, 0.01% sodiu...

Embodiment 2

[0060] In this example, the same kit as in Example 1 was used to conduct a parallel experiment of multiple samples in a 48-well plate at the same time.

[0061] This embodiment simultaneously extracts 8 simulated samples (artificially adding a concentration of 100ng / mL200bp fragment DNA to simulate free DNA in the blood), including the following steps:

[0062] Step 1): Add 200 μL of blood sample, 20 μL of proteinase K solution and 200 μL of DNA fixation binding solution to the 48-well plate with a multi-channel automatic pipette, parallel 8 samples, vortex for 30 seconds to mix well, and place in a water bath for digestion 10 minutes. The vibration frequency of the vortex mixer is set to 1150rpm;

[0063] Step 2): Take out the 48-well plate, add 50 μL of the first magnetic bead dispersion and 400 μL of isopropanol to the 8 sample wells with a multi-channel automatic pipette, and place the 48-well plate in a vortex mixer for 5 1 minute, place the 48-well plate on the 48-well...

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Abstract

The invention relates to a magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and an application method thereof. The kit comprises a DNA fixation combination solution, a first magnetic bead suspension, a cleaning solution, a genome DNA removal solution, a second magnetic bead suspension and an elution solution which are respectively packaged independently. The kit can be used for directly extracting free DNAs from the whole blood sample. The object for extracting and purifying free DNAs is widened from the serum or plasma to the whole blood sample, and the free DNA extraction step is extended forward to the blood preliminary treatment, thereby greatly reducing the original steps for extracting free DNAs from blood. Besides, the kit can also be used for extracting and purifying free DNAs from a plasma or serum sample. The kit can be used for removing genome DNA interference. The kit can be used for removing trace genome DNAs mixed in the whole blood, plasma or serum, thereby lowering the subsequent testing interference. Different reagents in the kit are compounded, so that the free DNA extraction efficiency and purity are maximally ensured.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a kit for extracting free DNA from whole blood based on a magnetic bead method and an application method thereof. Background technique [0002] DNA is the carrier of genetic information and one of the main research objects of molecular biology. With the deepening of molecular biology research and the rapid development of gene sequencing technology, the time and cost of gene sequencing have dropped rapidly. At the gene level Diagnosis of diseases has become a basic technical means of clinical diagnosis, and precision medicine based on gene sequencing is gradually becoming an important method in the fields of major disease diagnosis, personalized medicine, fetal genetic analysis, and individual identification. [0003] After the division of normal cells, tumor cells or fetal tissue cells, the DNA in the cells will be released into the body fluids and free from the cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2563/143C12Q2563/149
Inventor 吴巧曲峰尚春庆
Owner SUZHOU ENRICHING BIOTECH CO LTD
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