High-separation-degree detection method for content of plasmid super-spiral DNA

A detection method and supercoil technology, which are used in material separation, measuring device, analyzing materials, etc.

Pending Publication Date: 2020-09-29
南京济群生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The invention provides a high-resolution detection method to solve the problem of effectively separating supercoiled plasmid DNA from other topologically configured plasmid DNAs. Other topologically configured plasmid DNAs mainly include open-circle plasmid DNA, linear plasmid DNA, and concatemers At the same time, the homogenization degree of the supercoiled plasmid structure can be judged according to the peak shape of the supercoiled DNA separation, and the quality of the supercoiled DNA can be further evaluated

Method used

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  • High-separation-degree detection method for content of plasmid super-spiral DNA
  • High-separation-degree detection method for content of plasmid super-spiral DNA
  • High-separation-degree detection method for content of plasmid super-spiral DNA

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Experimental program
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Effect test

Embodiment 1

[0035] (1) Instrument and chromatographic conditions

[0036] High performance liquid chromatography: Agilent 1260infinity high performance liquid chromatography system and workstation;

[0037] Chromatographic column: TSKgel DNA-NPR (4.6mm×75mm, 2.5μm) polymethacrylate chromatographic column;

[0038] Prepare a 0.02mol / L Tris-HCl solution, adjust the pH to 9.0 with concentrated HCl as the mobile phase A, prepare a mixed solution with Tris-HCl and NaCl concentrations of 0.02mol / L and 1mol / L, and adjust the pH to 9.0 with concentrated HCl It is the mobile phase B phase, the proportion of phase A-B in the mobile phase is according to the time points of 0, 20, 25, and 30 minutes, the volume ratio of phase B is 50%, 76%, 50%, and 50%, and the set flow rate is 1.0mL / min, the detection wavelength is 260nm, and the column temperature is 40°C.

[0039](2) Experimental steps

[0040] Take an appropriate amount of plasmid standard (juventas, lot: 49443) with a size of 3.9 KB, and af...

Embodiment 2

[0046] (1) Instrument and chromatographic conditions

[0047] High performance liquid chromatography: Agilent 1260infinity high performance liquid chromatography system and workstation;

[0048] Chromatographic column: TSKgel DNA-NPR (4.6mm×75mm, 2.5μm) polymethacrylate chromatographic column;

[0049] Prepare a 0.02mol / L Tris-HCl solution, adjust the pH to 9.0 with concentrated HCl as the mobile phase A, prepare a mixed solution with Tris-HCl and NaCl concentrations of 0.02mol / L and 1mol / L, and adjust the pH to 9.0 with concentrated HCl It is the mobile phase B phase, the ratio of phase A to phase B in the mobile phase is according to the time points of 0, 20, 25, and 30 minutes, the volume ratio of phase B is 50%, 76%, 50%, and 50%, and the set flow rate is 0.8mL / min, the detection wavelength is 260nm, and the column temperature is 30°C.

[0050] (2) Experimental steps

[0051] Take an appropriate amount of plasmid standard (juventas, lot: 49443) with a size of 3.9 KB, a...

Embodiment 3

[0056] (1) Instrument and chromatographic conditions

[0057] High performance liquid chromatography: Agilent 1260infinity high performance liquid chromatography system and workstation;

[0058] Chromatographic column: TSKgel DNA-NPR (4.6mm×75mm, 2.5μm) polymethacrylate chromatographic column;

[0059] Prepare a 0.02mol / L Tris-HCl solution, adjust the pH to 9.0 with concentrated HCl as the mobile phase A, prepare a mixed solution with Tris-HCl and NaCl concentrations of 0.02mol / L and 1mol / L, and adjust the pH to 9.0 with concentrated HCl It is the mobile phase B phase, the proportion of phase A-B in the mobile phase is according to the time points of 0, 20, 25, and 30 minutes, the volume ratio of phase B is 50%, 76%, 50%, and 50%, and the set flow rate is 1.0mL / min, the detection wavelength is 260nm, and the column temperature is 40°C.

[0060] (2) Experimental steps

[0061] Take an appropriate amount of plasmid standard product (lot:20190510) with a size of 3.9KB, and af...

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Abstract

The invention relates to the technical field of gene therapy drug analysis, in particular to a high-separation-degree detection method for the content of plasmid superspiral DNA. The invention discloses an analysis method for simultaneously separating four topological structures of plasmid DNA (deoxyribonucleic acid) and evaluating the homogenization condition of superspiral DNA. The analysis method comprises the following steps: preparing an analysis solution; with an anion exchange chromatographic column and a mixed solution of Tris-HCl-NaCl as a mobile phase, using a gradient elution method; and measuring the prepared analysis solution on a machine. According to the analysis method, four topological structures of plasmid DNA can be effectively and accurately separated and determined, the homogenization condition of the super-helix DNA can be evaluated, the stability of the super-helix DNA is further evaluated, so that the index quality of a product is accurately detected.

Description

technical field [0001] The invention relates to the technical field of nucleic acid drug analysis, in particular to an IEX-HPLC high-resolution detection method for the supercoiled DNA content of a plasmid. Background technique [0002] Plasmids are small DNA molecules that can replicate autonomously. The artificially modified engineered plasmids can be used as a drug to express the inserted target gene in the patient's body, and finally achieve the purpose of preventing or treating related diseases. At present, plasmid DNA has become the representative of the next generation of biopharmaceuticals and is intended to be used in gene vaccines and gene therapy. With the surge of clinical research, the demand for plasmid DNA has also greatly increased, which also puts forward requirements on the quality of plasmid DNA. [0003] Like other oligonucleotides, plasmids are very sensitive to nucleases and physical shearing forces, and DNA degradation will directly affect the quality...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34
CPCG01N30/02G01N30/06G01N30/34
Inventor 苏月焱芮勉文谭颖茹万瑶瑶罗琦田超赵卿
Owner 南京济群生物科技有限公司
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