Process for separating nucleic acid from biological particles by solid-phase carrier

A technology of biological particles and solid-phase carriers, applied in the fields of sugar derivatives, organic chemistry, fermentation, etc., can solve the problems of many steps and difficult miniaturization

Inactive Publication Date: 2002-06-26
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When separating high-molecular DNA, samples often need to be pre-treated, so there are many steps and it is difficult to achieve miniaturization

Method used

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  • Process for separating nucleic acid from biological particles by solid-phase carrier
  • Process for separating nucleic acid from biological particles by solid-phase carrier
  • Process for separating nucleic acid from biological particles by solid-phase carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Separation of macromolecular DNA. Take the adsorption of DNA fragments with nano-magnetic microspheres as an example: add 40 μl of 15 mg / ml nano-magnetic microsphere suspension and 0.4 μg / μl double-stranded DNA molecular weight marker (marker ) (Hind III digestion) 15μl, shake gently on the vortex shaker for 15s, then add 4mol L -1 80μl of NaI solution, after shaking slightly for 15s, let stand for 3 minutes. Then add 100 μl of isopropanol as a suction aid, shake slightly for 5 seconds, and then let stand for 2 minutes. The magnetic spheres were fixed with a magnetic separation rack, the supernatant was discarded, and the nanomagnetic spheres that had adsorbed DNA were washed twice with 100 μl of 70% ethanol. Add 50 μl of TE solution (10mol L -1 Tris.Cl, 1mmol L -1 EDTA). The DNA was eluted from the magnetic beads in a water bath at 62°C for 12 minutes. Then the magnetic ball is separated from the TE solution, and the obtained TE solution is subjected t...

Embodiment 2

[0023] Example 2: Separation of genomic (genomic) DNA of Escherichia coli with nano-magnetic microspheres.

[0024] The samples were plasmid-free E. coli strains cultured overnight. Take 300 μl of the harvested cell culture solution, and then add 50 μl of nano-magnetic microsphere suspension. After shaking and mixing, use a magnetic separation rack to separate the magnetic beads, and discard the supernatant. Add 300 μl cell lysate (3mol / l NaI, 4mol / l urea, 30g / l Triton-100 (trinitrotoluene), 10mmol / l EDTA (pH6.5), 25mmol / l Tris·HCl (pH6.5) to the magnetic ball .5)) (the same below), shake well, and let stand at room temperature for 5 minutes. Add 300 μl of isopropanol. Slightly oscillate on a vortex shaker for 15 s, and let stand at room temperature for 5 min. Fix the magnetic beads with a magnetic separation rack, discard the supernatant, and wash the magnetic beads twice with 70% ethanol. Then add the above 100 μl TE solution (pH=6.0), and let it stand at room temperatu...

Embodiment 3

[0025] Example 3: Adsorption and separation of leukocytes in whole blood by nano-magnetic microspheres and extraction of DNA therein.

[0026] Whole blood used in this experiment was provided by healthy blood donors, and anticoagulated with 1 / 6 volume of blood ACD (23mmol / l citric acid, 80mmol / l glucose, 45mmol / l sodium citrate). Take an appropriate amount of 300 μl of blood, add 50 μl of the above-mentioned magnetic microsphere solution (suspended with the above pH=6.0 TE solution), oscillate slightly on a vortex shaker for 15 s, and after standing at room temperature for 3 min, fix the magnetic beads with a magnetic separation rack. Discard other parts. Add 300 μl of cell lysate, shake and mix well, and let stand at room temperature for 2 minutes. Then add 300 μl isopropanol, shake and mix well, and let stand at room temperature for 5 minutes. Fix the magnetic beads with a magnetic separation rack and discard the supernatant. After washing the magnetic ball twice with 70%...

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Abstract

A process for using solid-phase carrier to separate nucleic acid from biologic particles features that the nm-class magnetic microspheres emulsion or the specially treated fibrous membrane is used as solid-phase adsorbing medium to separate cell from biologic particle, the cell-cracking liquid is used to crack cell structure, the solid-phase adsorption and desorption are used to directly collect the biologic particles rich in nucleic acid from biologic specimen such as blood plasma, etc., and the nucleic acid is extracted. Its advantages include simple process, high speed, and easy autoamtization and miniaturization.

Description

technical field [0001] A method for separating nucleic acid from biological particles by using a solid phase carrier belongs to the technical field of rapidly separating and lysing cells of biological particles to obtain nucleic acids. Background technique [0002] Based on the biochip, the "miniature biochip laboratory" that integrates sample processing, biological reaction and result detection on the microarray technology platform is an advanced form of biochip technology. Currently, the chip construction of biochemical reaction and result detection devices Great progress has been made, but how to build an automated sample preparation device on a chip is still a difficult problem. This is because: biological particles such as leukocytes, virus particles, epithelial cells, cultured cells and other biological particles containing nucleic acid, protein and other target molecules are enriched from biological samples such as whole blood, plasma, saliva, urine, cell and tissue c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C12P19/34
Inventor 张旭谢欣陈继明陈德朴费维扬程京
Owner TSINGHUA UNIV
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