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Method for measuring content of superhelix DNA

A determination method and supercoiled technology, applied in the biological field, can solve the problems of cumbersome sample processing, easy to cause errors, unsuitable and other problems

Inactive Publication Date: 2014-06-11
HUMANWELL HEALTHCARE GRP
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Problems solved by technology

However, plasmid DNA is not only mixed with impurities such as RNA and protein, but also contains a small amount of open-circle DNA, which interferes with the supercoiled DNA chromatographic peak during the actual detection process and affects the final quantitative detection.
The capillary gel electrophoresis or agarose gel electrophoresis methods used in the existing technology for detecting supercoiled DNA content have shortcomings in detection methods. For example, capillary gel electrophoresis requires tedious sample processing and is not suitable for production of plasmid DNA. used in each step; agarose gel electrophoresis at OD 260 The detection range is too narrow under the wavelength, and it is easy to cause errors in the sample dilution process, the accuracy is very low, and it is not suitable for use in every step of plasmid DNA production
More importantly, neither of the above two methods can quantitatively detect the content of supercoiled DNA

Method used

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  • Method for measuring content of superhelix DNA
  • Method for measuring content of superhelix DNA
  • Method for measuring content of superhelix DNA

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Experimental program
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Effect test

Embodiment

[0042] 1. Pretreatment

[0043] Take 1g of Escherichia coli and place it in 10ml Buffer1 (50mM Tris, 50mM glucose, 10mM EDTA, pH7.5) to suspend. After mixing well, slowly add 10ml Buffer2 (0.2M NaOH, 1%SDS), and stir gently. Form a homogeneous gel. Then slowly add 10ml Buffer3 (3M potassium acetate, pH 5.5), stir gently until completely neutralized, and let stand at 4°C for 1h. Use gauze to filter to remove the white precipitate in the solution, and then filter with 0.22um gauze, and the filtrate is used for chromatographic column testing.

[0044] 2. Source of standard plasmid and preparation of standard curve

[0045] The source of the standard plasmid: pUDK-HGF plasmid produced by ourselves. The supernatant of the E. coli lysate was purified by Sepharose6Fast Flow, Plasmind Select Xtra, and Source30Q, and the purity was over 99% by HPLC.

[0046] Preparation of standard curve:

[0047] Take 30ml of the purified plasmid and add 70ml of absolute ethanol, mix well and set...

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Abstract

The invention discloses a method for measuring content of superhelix DNA. The method comprises the following steps: performing first detection and separation treatment on a sample to be measured by adopting an AKTAexplorerTM10 detection instrument and utilizing a molecular sieve chromatography column so as to obtain total DNA samples, and calculating the mass content of the total DNA samples; and performing second detection and separation treatment on the total DNA samples by adopting a thiophilic aromatic chromatographic column, and calculating to obtain the mass content of the superhelix DNA in the sample to be measured. According to the method, the content of the superhelix DNA in plasmid DNA can be quantitatively and effectively measured, the analytical accuracy is equivalent to that of capillary gel electrophoresis, and the method is obviously superior to agarose gel electrophoresis and suitable for each step in DNA production.

Description

technical field [0001] The invention relates to the field of biology, in particular, the invention relates to a method for determining the content of supercoiled DNA. Background technique [0002] In the industrial production of plasmid biological products, supercoiled DNA is the final target product. However, plasmid DNA is not only mixed with impurities such as RNA and protein, but also contains a small amount of open-circle DNA, which interferes with the supercoiled DNA chromatographic peak during the actual detection process and affects the final quantitative detection. The capillary gel electrophoresis or agarose gel electrophoresis methods used in the existing technology for detecting supercoiled DNA content have shortcomings in detection methods. For example, capillary gel electrophoresis requires tedious sample processing and is not suitable for production of plasmid DNA. used in each step; agarose gel electrophoresis at OD 260 The detection range under the wavelen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 王学海李杰李莉娥许勇何昆陈爱芳吕兴凯田吕明马梵辛杨仲文涂荣华肖强张绪文马铮裴达黄韫韬
Owner HUMANWELL HEALTHCARE GRP
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