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Method for fixing a supercoiled DNA and the use for analysing the dna repair

A supercoiled, porous polymer technology, used in biochemical equipment and methods, transportation and packaging, and microbial assay/inspection, etc., can solve problems such as DNA damage, unpredictable covalent coupling, and plasmid fragmentation

Active Publication Date: 2008-08-27
COMMISSARIAT A LENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0023] Covalent coupling of DNA to vectors is unpredictable due to the stress it creates causing breakage on the plasmid
Furthermore, it includes chemical and / or physical treatment steps (heating, UV-irradiation), which can be damaging to DNA

Method used

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  • Method for fixing a supercoiled DNA and the use for analysing the dna repair
  • Method for fixing a supercoiled DNA and the use for analysing the dna repair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0065] Example : Application of DNA Immobilized in Hydrogels as a Substrate for Analyzing DNA Repair

[0066] The principle of a DNA repair assay for comparing the repair of DNA immobilized on poly-L-lysine-coated or hydrogel-coated supports is described in patent application FR 0216435.

[0067]1) Materials and methods

[0068] a) Preparation of damaged supercoiled DNA

[0069] Plasmids (pBluescript II, Stratagene) were produced by transformation of the XL1 blue MRF chain of E. coli (Stratagene) according to the protocol provided by the supplier. Plasmid using Plasmidmidi kit TM (Qiagen) and diluted in PBS buffer (10 mM phosphate, pH 7.4, 137 mM NaCl, 2 mM KCl).

[0070] Plasmids are treated with different physical or chemical agents to introduce damage therein, namely:

[0071] -UVC-irradiation (UVC)

[0072] Plasmid solutions (25 μl; 40 μg / ml) in PBS were illuminated using a lamp emitting at 254 nm. The received dose is 0.3J / cm 2 (Figure 1 and 2) or 0.03, 0.06 an...

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Abstract

A method for fixing a supercoiled DNA consists in deposing a supercoiled DNA sample on the surface of a porous polymer film, in fixing said supercoiled DNA therein by a passive diffusion, in obtaining a support by the inventive method and in using said support for analysing the DNA distribution.

Description

technical field [0001] The present invention relates to a method for immobilizing supercoiled DNA and the use of supercoiled DNA immobilized on a carrier as a substrate for the analysis of enzymatic reactions for DNA repair. Background technique [0002] Biochips have become an essential analytical tool for biomedical research and clinical diagnosis. These powerful tools can simultaneously analyze tens of thousands to hundreds of thousands of samples with the aid of DNA probes (oligonucleotides, cDNA, PCR amplification products) or protein probes (antibodies, peptides) immobilized on carriers. [0003] Biochips are prepared using a support (glass, polypropylene, polystyrene, silicone, metal, nitrocellulose, or nylon) that is optionally covered with a porous membrane [nylon-coated glass (Atlasarrays TM ; Clontech); silicone resin coated with hydrogel (Nanochip TM ; Nanogen), coated with acrylamide polymer gel Hydrogel TM (Perkin-Elmer) or glass (US application 2005 / 0042363...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/68Y10T428/31623C12Q2525/307C12Q1/6834C12Q1/6837
Inventor J-F·米约S·索瓦热
Owner COMMISSARIAT A LENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES
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