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A rapid method for detecting the damage intensity of DNA fragmentation caused by plasma in vitro

A plasma and intensity technology, applied in measuring devices, material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of inaccurate measurement of ARTP dose, lack of rapid detection method of DNA breakage damage intensity, complicated operation, etc.

Active Publication Date: 2020-10-27
WUXI TMAXTREE BIOTECHNOLOGY CO LTD +1
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Problems solved by technology

For the operating parameters of the ARTP mutagenesis system, power is usually used as a parameter, and power (P in )20-120W, gas flow (Q He )5-20slpm, irradiation distance (D) is 2-5mm, but the dose of ARTP has not been accurately measured under different operating conditions
[0007] The R&D team has qualitatively verified the damage of ARTP to DNA by means of gel electrophoresis, but so far, there is a lack of rapid detection methods for the intensity of DNA damage, which can then be used to characterize the dose of ARTP
Comet method (single-cell gel electrophoresis) is a semi-quantitative method for detecting DNA breakage damage (Kumari S, Rastogi R P, Singh KL, et al. DNA damage: Detection strategies. Excli Journal, 2008, 7:44-62. ), but its operation is complicated and can only be used for large DNA fragments

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  • A rapid method for detecting the damage intensity of DNA fragmentation caused by plasma in vitro
  • A rapid method for detecting the damage intensity of DNA fragmentation caused by plasma in vitro
  • A rapid method for detecting the damage intensity of DNA fragmentation caused by plasma in vitro

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Embodiment Construction

[0034] The method of the present invention will be described by taking Atmospheric and Room Temperature Plasma (ARTP) in the plasma as an example.

[0035] figure 1 It is a schematic diagram for determining the damage strength of ARTP on DNA fragmentation in vitro. Firstly, ARTP is used to treat DNA-coated microspheres, and the active particles produced by ARTP react with the DNA on the microspheres through reaction or energy transfer, and randomly break the DNA chains on the microspheres. DNA fragments with different lengths after being broken from different sites are detached from the microspheres and free in the aqueous solution outside the microspheres. The degraded DNA fragments can be separated from the treated DNA microspheres by centrifugation. SYBRGreen is used to stain the treated DNA-coated microspheres, and the amount of remaining DNA on the microspheres can be determined by fluorescence spectrophotometry. Compared with the amount of DNA on the microspheres bef...

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Abstract

The invention relates to a method for rapid detection of in vitro DNA break damage strength by plasma, and the method is used for determining the damage strength of plasma mutagenesis methods including atmospheric and room temperature plasma (ARTP) on DNA. The method includes the steps of: 1) treating DNA coated microspheres by plasma mutagenesis method, and setting a control group without plasma treatment; 2) performing washing, and then dyeing the DNA coated microspheres with a dye; 3) determining the fluorescence value of the dyed DNA coated microspheres by a fluorescence detector; and 4) calculating the DNA relative content of the control group and the plasma mutagenesis treated microspheres according to the fluorescence value, and judging the gene damage strength of plasma. The invention utilizes DNA coated microspheres to establish a rapid detection method for in vitro DNA damage strength so as to realize direct and effective evaluation of plasma caused damage strength to DNA.

Description

technical field [0001] The invention relates to a method for rapid detection of DNA breakage damage intensity in vitro by plasma, in particular to the establishment of a rapid detection method for in vitro DNA damage intensity by using DNA-coated microspheres. Background technique [0002] High-quality microbial strains are always the core of the biological industry. How to use efficient biological mutagenesis technology to achieve rapid optimization of strains is an important task in the biological industry. [0003] By means of physical (such as X-ray, γ-ray, UV, ion beam implantation, plasma), chemical (such as alkylating agent, azide), biological (such as expressing mutS, ndk gene) and other means to improve the mutation rate, it can be Create more genotypes in a short time. Different mutagenesis methods cause DNA damage in different ways, such as ultraviolet (UV) mainly causing cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PPs), resulting in C to T muta...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 邢新会张翀张雪王立言
Owner WUXI TMAXTREE BIOTECHNOLOGY CO LTD
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