Method for extracting and purifying nucleic acid from samples by magnetic beads

A magnetic bead and nucleic acid technology, applied in the field of nucleic acid extraction and purification, can solve the problems of unfavorable identification molecule activity preservation, low adsorption efficiency, narrow application range, etc., and achieve the effects of saving manpower, fast operation, and convenient transportation

Active Publication Date: 2010-03-10
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] These magnetic beads modified by active recognition molecules such as streptavidin, biotin, antibodies or oligonucleotide fragments are not conducive to the preservation of the activity of the recognition molecules during storage and transportation, the use range is narrow, the adsorption efficiency is not high, and the price is extremely high. Not widely used due to high cost

Method used

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  • Method for extracting and purifying nucleic acid from samples by magnetic beads
  • Method for extracting and purifying nucleic acid from samples by magnetic beads
  • Method for extracting and purifying nucleic acid from samples by magnetic beads

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Separation and purification of HBV DNA in serum by peptide-oligonucleotide magnetic beads

[0052] The present embodiment comprises the following steps: (1) peptide-oligonucleotide modified magnetic beads: with γFe 2 o 3 and Fe 3 o 4 The homogeneous, superparamagnetic, and single-divergent polymeric microspheres synthesized by magnetic materials are used as the magnetic source. Each microsphere is coated with a layer of polystyrene, and the magnetic polymer carrying carboxylate is prepared by emulsion polymerization or dispersion polymerization. Microspheres; carboxylate-modified polystyrene core particles coated with a magnet, oligo(dT) 14 Then covalently bonded to this surface, nucleic acid molecules can be well anchored on this oligonucleotide [see Figure 1 (a)]; (2) Cell lysis: add 200 μl of HBV DNA positive serum to a 1.5ml centrifuge tube , followed by adding 300 μl of lysate, the lysate is composed of 0.5% (W / V) SDS, 2% (V / V) Triton X-100, 1mol / L G...

Embodiment 2

[0054] Example 2: Separation and purification of HBV DNA in serum and detection by fluorescent quantitative PCR

[0055] According to the method described in Example 1, after adsorption and washing, use a pipette to take 50 μl of PCR reaction solution and repeat several times to completely elute the magnetic beads adsorbed on the wall of the centrifuge tube, transfer them to the PCR reaction tube, and use fluorescent quantitative PCR instrument for quantitative detection.

[0056] 1) Three concentrations of HBV DNA-positive serum calibrated by the China Institute for the Control of Biological Products (National Institute for the Control of Hepatitis B Virus) nucleic acid quantitative standards were used as samples to be tested, and the concentrations were 5.0×10 6 IU / ml, 5.0×10 4 IU / ml, 5.0×10 2 IU / ml, the experiment was repeated 8 times for each concentration, and the results are shown in the table below.

[0057]

[0058] Such as figure 2 As shown, using the method of...

Embodiment 3

[0065]Embodiment 3: The peptide-oligonucleotide magnetic bead HBV DNA extraction of the present invention compares with the magnetic bead method existing on the market to separate and purify nucleic acid comparison

[0066] Using magnetic beads with three different treatment methods and the magnetic beads treated with peptide-oligonucleotide of the present invention to separate and purify HBV DNA in serum and perform fluorescence quantitative PCR detection comparison. The three kinds of magnetic beads are: Invetrogen Dynal magnetic beads, Carboxylate (carboxylate) speed magnetic beads, and Carboxylate magnetic beads.

[0067] The HBV-positive serum calibrated with the Hepatitis B virus (HBV) nucleic acid quantitative standard of the China National Institute of Inspection and Quarantine was used as the initial sample, and the gradient was diluted to 10 5 IU / ml, 10 4 IU / ml, 10 3 IU / ml, 10 2 IU / ml, 10IU / ml, 5 positive sera and one negative sera are used as samples to be tested...

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Abstract

The invention relates to a method for extracting and purifying nucleic acid from samples by magnetic beads, which comprises the following steps: (1) magnetic bead treatment: modifying the magnetic beads by peptide-oligonucleotide; (2) cell lysis: adding the sample containing target nucleic acid and needing to be separated into a centrifuge tube, and then adding lysis buffer to lyse the cells; (3)nucleic acid adsorption: adding the magnetic beads modified by the peptide-oligonucleotide into the solution to lead the nucleic acid to be adsorbed on the surfaces of the magnetic beads; placing thecentrifuge tube into a magnetism separation machine to lead the magnetic beads to be adsorbed on the tube side; (4) impurities removal: adding aqueous phase buffer solution to wash the magnetic bead which are adsorbed with the nucleic acid on the surfaces and adsorbed on the tube side so as to separate other impurities on the magnetic bead; and (5) nucleic acid recovery: directly taking the nucleic acid adsorbed on the surfaces of the magnetic beads as the template for PCR amplification, or adding elution buffer to lead the nucleic acid molecules adsorbed on the surfaces of the magnetic beadsto be released into the buffer for downstream experiments. The magnetic beads used by the method can be reserved at the temperature of 4 DEG C or -20 DEG C, and have convenient transport, high adsorption efficient and lower cost. The nucleic acid extracted and purified by the method has wide application range.

Description

technical field [0001] The invention relates to a method for extracting and purifying nucleic acid from samples by using magnetic beads. Background technique [0002] Nucleic acid extraction and purification technology is a basic technology of biochemistry and molecular biology. As molecular biology techniques are widely used in biology, medicine and related fields, nucleic acid extraction and purification techniques have also been further developed. [0003] Nucleic acid is always combined with various proteins in cells. Nucleic acid extraction mainly refers to the separation of nucleic acid from biological macromolecules such as proteins, polysaccharides, and fats. Most nucleic acid extraction methods generally include cell lysis, removal of biological macromolecules such as proteins, polysaccharides, and lipids bound to nucleic acids, removal of other unnecessary nucleic acid molecules, precipitation of nucleic acids, removal of impurities such as salts and organic solv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07H21/00C12Q1/68C12Q1/70
Inventor 戴立忠熊晓燕
Owner SANSURE BIOTECH INC
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