CN102146455A - Cell DNA damage detection kit and detection method thereof - Google Patents
Cell DNA damage detection kit and detection method thereof Download PDFInfo
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Abstract
The invention provides a cell DNA damage detection kit and a detection method thereof, relating to the cell DNA damage detection technique. The cell DNA damage detection kit comprises low melting-point agarose, a cell lysis solution, a cell despiralization solution and a DNA staining agent. The detection method comprises the following steps of: evenly mixing the cell DNA damage sample suspension with the low melting-point agarose gel, spotting the mixed liquid into sample holes on a gel board; immersing the gel board into the cell lysis solution for lysis in a dark place; immersing the gel board after lysis into the cell despiralization solution for despiralization, performing electrophoresis after despiralization; and dropwise adding the DNA staining agent for staining after electrophoresis, and analyzing the detection result under a fluorescence microscope. According to the detection method, the board cover of a cell culture board with 96 holes is used as the gel board to substitute for a glass slide for performing single cell gel electrophoresis, and compared with the traditional process in which 2 or 3 layers of gel are laid on a sample glass slide for performing single cell gel electrophoresis, two steps of gel laying and spotting are combined into one step, so that the sample preparation process is simple and easy to operate, the gel can not fall off and the success rate is high.
Description
Technical field
The present invention relates to cell DNA damage check technology, particularly a kind of cell DNA injury testing reagent box and detection method thereof.
Background technology
DNA is a kind of important biomacromolecule that has the genetic information transfer function in the cell, is the important research object of Biochemistry and Molecular Biology.Dna molecular constitutes double-spiral structure by antiparallel two polynucleotide chains via basepairing rule, its entrained genetic information is by kind, quantity and the ordering decision of the base on the polynucleotide chain, base sequence is hidden in the inside of dna double spiral deeply, and the double-spiral structure of two polynucleotide chain compositions has formed stable dna molecular and it can correctly be conveyed hereditary information.
In the biological living environment the multiple factor is arranged, as physical agent ionizing rays, ultraviolet ray, electromagnetic radiation etc.; Chemokines oxynitride, coal smoke, vehicle exhaust etc.; Biotic factor bacterium, virus etc.; They can both cause the dna damage of cell.The form of dna damage has formation, dna adduct, point mutation, the DNA of DNA chain break, pyrimidine dimer crosslinked etc.The dna damage that environmental factor causes may influence the genetic stability of cell, and may exert an influence to the offspring, as cause miscarriage, stillborn foetus, monster and some heredopathia, also can cause tumour generation (1, Fairbairn D W, Olive P L, O ' Neill K L The Comet assay:a comprehensive review, Mutation Research.1995,339 (1), 37-59; 2, thank into English .DNA damage and tumorigenic progress, " Chinese cancer magazine ", 2006,16 (4), 313-317.).Because the dna damage of cell can cause disease, threaten human and animal's health, so the pair cell dna damage detects, and study its mechanism of action and just seem very important.
At present, the detection method of general in the world dna damage can be divided into two classes: the one, and the biological effect that causes from damage detects, as micronucleus test, chromosome aberration, sister chromosome exchange test etc.The 2nd, directly detect dna damage, as elution technique, sedimentation techniques, single cell gel electrophoresis detection etc.Single cell gel electrophoresis (single cell gel electrophoresis, SCGE), also claim comet experiment (comet assay), be meant unicellular level carry out dna damage and the fluorescence detection method that repair to detect (3, Liao W, McNutt MA, Zhu WG The comet assay:a sensitive method for detecting DNAdamage in individual cells.Methods.2009,48 (1): 46-53.), this method has simply, fast, characteristics such as sensitivity, original position.Cell through cracking, untwist, behind the electrophoresis, the DNA of fracture can shift out in examining under the effect of electrical forces, behind the fluorescent dyeing, under fluorescent microscope, can be observed the comet formation image of damaged cell nuclear, present one and have the bright comet head and the comet afterbody of a disperse.Can judge dna damage degree on the unicellular level by this image being measured with the computer graphic conformal analysis.At present single cell gel electrophoresis be considered to study low dosage genetic toxicity effect effective tool (4, Sirota NP, Kuznetsova EA.The comet assay application in radiobiological investigations.Radiats Biol Radioecol.2010,50 (3): 329-339.).
Single cell gel electrophoresis technique is to develop on nuclear precipitation technology basis, what very early time adopted is neutral microgel technology, yet cracking and electrophoresis can only detect the double-stranded DNA part under neutrallty condition, can not measure the strand part, and a lot of genetoxic material inductive dna single chain fragment quantity is higher 5～2000 times than double-stranded part.SinghNP (5, Singh N P, McCoyMT, Tice R R et al.A simple technique for the puantitation of low levels of DNA damage in individual cells.Exp.Cell Res.1988,175:184-191.) etc. alkaline electrophoretic technique proposed first, can detect the single stranded DNA part, single cell gel electrophoresis susceptibility is improved greatly.When pH＞13, except dna double chain and single-strand break, can also detect the alkaline unstable structure of DNA.The comet experiment of standard is difficult for detecting the crosslinked phenomenon of DNA, comprise that DNA-protein-crosslinking and DNA-DNA are crosslinked, if but when handling cell, added linking agent and DNA splitting of chain agent simultaneously, the DNA crosslinked action that chemical substance causes just could be detected indirectly by measuring dna break.Through updating, the comet experimental technique has obtained important development and application, and its advantage can be summarized as: the heterogeneous information that 1) individual cells DNA can be provided; 2) be applicable to all eukaryotic cells basically; 3) sensitive more, can check 0.1DNA/109 dalton fracture; 4) required cell sample amount little (＜10000); 5) need not radioactive tracer etc.Based on this, at present, the dna damage that single cell gel electrophoresis has been widely used for karyocyte with repair research, as genotoxicity detection and molecular epidemiology, genetic toxicology, tumor invasion mechanism and Clinics and Practices, environmental organism monitoring, aging and Apoptosis Mechanism, radiobiology, drug screening etc.
At present, the basic step that is used for the specimen preparation of single cell gel electrophoresis analysis comprise (6, Cemeli E, BaumgartnerA, Anderson D.Antioxidants and the Comet assay.Mutat Res.2009,681 (1): 51-67.): preparation, the lysis of sample gel slide glass, untwist, electrophoresis, neutralization washing, fluorescent dye.Associated picture software analysis fluoroscopic image can be shot with video-corder and utilize to the sample slide for preparing under fluorescent microscope, the degree of injury of DNA is estimated.The laboratory method complex steps of tradition single cell gel electrophoresis, need configuration lytic reagent separately, need in advance sample DNA to be handled, experimental period is long, and each test sample is limited, and the gel slide will be spread 2 layers or 3 layers of gel, and manufacture craft is more loaded down with trivial details, and easy flake causes the failure of an experiment in later stage electrophoresis and washing, has influenced the promotion and application of single cell gel electrophoresis.
Summary of the invention
The object of the present invention is to provide a kind of cell DNA injury testing reagent box.
Another object of the present invention is to provide a kind of fast and convenient, cell DNA damage detecting method that technology is simple, highly sensitive based on single cell gel electrophoresis technique.
Described a kind of cell DNA injury testing reagent box is provided with box body and reagent, and described reagent comprises that low melting-point agarose, cell pyrolysis liquid, cell separate spinning liquid and DNA staining agent.
Described cell pyrolysis liquid is made up of solution A and solution B, and the prescription of described solution A is: 2～3mol/LNaCl, 0.1～0.2mol/L EDTA-Na
2, 5～100mmol/L Tris, massfraction are 0.5%～1.5% sodium sarcosinate, surplus is deionized water; The prescription of described solution B is: volume fraction is 1%～2%Triton-100, and 5%～15% dimethyl sulfoxide (DMSO) (Dimethyl sulfoxid, DMSO), surplus is deionized water; Described solution A was mixed with solution B and is promptly got cell pyrolysis liquid in 1: 1 by volume; The prescription that described cell is separated spinning liquid is: 0.2～0.5mol/LNaOH, 1～2mmol/LEDTA-Na
2, surplus is deionized water; Described DNA staining agent be propidium iodide (Propidium Iodide, PI), ethidium bromide (EB), Hoechst 33342, hoechst33358 or DAPI etc.
The screening formulation of described solution A is: 2.5mol/LNaCl, 0.1mol/LEDTA-Na
2, 10mmol/LTris (pH=10), massfraction are 1% sodium sarcosinate, surplus is deionized water.
The screening formulation of described solution B is: volume fraction is 1%Triton-100 and 10% dimethyl sulfoxide (DMSO), and surplus is deionized water.
The screening formulation that described cell is separated spinning liquid is: 0.3mol/L NaOH and 1mmol/L EDTA-Na
2(pH=13.0), surplus is deionized water.
The concentration of described propidium iodide can be 1～5 μ g/ml, and the concentration of described ethidium bromide can be 10～100 μ g/ml.
Described cell DNA damage detecting method may further comprise the steps:
1) cell DNA is damaged sample suspension and low melting-point agarose gel mixing gets mixed solution, again with the mixed solution point sample in the gel slab sample well;
2) gel slab behind the point sample is immersed lucifuge cracking in the cell pyrolysis liquid;
3) gel slab after the cracking is immersed cell and separate in the spinning liquid and untwist, the rear electrophoresis that finishes untwists;
4) the gel slab sample well behind the electrophoresis drips the dyeing of DNA staining agent, analyzing and testing result under fluorescent microscope.
In step 1), the massfraction of described low melting-point agarose gel can be 0.8%～1.5%, and the volume ratio of described cell DNA damage sample suspension and low melting-point agarose gel can be 1: (2～10); The volume of described point sample can be 10～25 μ l/ sample wells; Described gel slab can adopt the plate lid of 96 porocyte culture plates, and the depression that described plate covers can be used as sample well; Described concave bottom can be carved with cut.
In step 2) in, described cell pyrolysis liquid can carry out 4 ℃ of precoolings before using; Described cracked condition can be: 4～10 ℃ of temperature, time 1～1.5h; Available PBS detergent gel plate was 1～3 time after described cracking was finished, each 5～10min.
In step 3), described condition of untwisting can be: 4～10 ℃ of temperature, time 30～45min; Described electrophoretic condition can be: 4～10 ℃ of temperature, time 30～60min, electric current 50～150mA.Available Tris damping fluid detergent gel plate was 1～3 time after described electrophoresis was finished, each 5～10min.
In step 4), described painted condition can be: 4～10 ℃ of temperature, time 5～20min.
Compare with existing single cell gel electrophoresis technique, the present invention has following advantage:
1) the present invention can replace slide glass to be used for single cell gel electrophoresis as gel slab with the plate lid of 96 porocyte culture plates, will spread the technology of 2,3 layers of glue compares with the sample slide of traditional single cell gel electrophoresis, the present invention will spread glue point sample step and unite two into one, make that specimen preparation technology of the present invention is simple, operation easily, do not come unstuck the success ratio height.
2) the present invention can adopt the direct point sample of homemade gel slab, compares with traditional slide glass, and fluorescence decay reduces, and has improved fluorescent brightness, thereby has improved the sensitivity and the picture quality of experiment.
3) the invention provides the test kit that is used for cell DNA damage rapid detection, available its directly carries out the detection of DNA cell injury sample, and whole testing process can be finished at 2.5～3h, and is convenient and swift.
Description of drawings
Fig. 1 detects the shows fluorescent microscopy images of the single cell gel electrophoresis of people's liver cancer SMMC7721 cell for the test kit among employing the present invention.In Fig. 1, control is a blank, and used medicine is an etoposide, and its concentration is followed successively by 12.5 μ mol/L, 25 μ mol/L, 50 μ mol/L, and be 12h action time, and used cell is liver cancer SMMC7721.
Fig. 2 detects the shows fluorescent microscopy images of the single cell gel electrophoresis of people's liver cancer MHCC97-H cell for the test kit among employing the present invention.In Fig. 2, control is a blank, and used medicine is an etoposide, and its concentration is followed successively by 12.5 μ mol/L, 25 μ mol/L, 50 μ mol/L, and be 12h action time, and used cell is liver cancer MHCC97-H.
Fig. 3 is for adopting the shows fluorescent microscopy images of test kit of the present invention to the single cell gel electrophoresis of human liver cell Changliver.In Fig. 3, control is a blank, and used medicine is a neocarzinostatin, and activity is 10ng/ml, 100ng/ml, and be 12h action time, and used cell is liver cell Changliver.
Embodiment
The invention will be further described by the following examples, but embodiments of the present invention are not limited thereto.
The making of embodiment 1 gel slab
The used gel slab of the present invention needn't use slide glass, can use the common cell cultures in laboratory to replace slide glass to be used for single cell gel electrophoresis with the plate lid of 96 well culture plates, and each depression that 96 orifice plate plates cover all can be used for as sample well.Get one 96 orifice plate plate lid, dismiss bossing all around with sharp paper knife, quantity per sample is cut into sizeable gel slab, as being cut into the gel slab that contains 12,24,36,48,64,96 depressions, but each sample well cut makes that sepharose is difficult for coming unstuck.
The preparation of embodiment 2 cell DNA injury testing reagent boxes
Ordinary method is adopted in the configuration of reagent solution, and the test kit that can carry out 400 sample DNA damage check comprises:
1) 1g low melting-point agarose.
2) 150ml solution A and 150ml solution B.The prescription of solution A is: 2.0～3.0mol/L NaCl, 0.1～0.2mol/LEDTA-Na
2, 5～100mmol/L Tris, massfraction 0.5%～1.5% sodium sarcosinate, surplus is deionized water.The screening formulation of solution A is: 2.5mol/L NaCl, 0.1mol/L EDTA-Na
2, 10m mol/L Tris (pH=10.0), massfraction are 1.0% sodium sarcosinate, surplus is deionized water.
The prescription of described solution B is: volume fraction is 1.0%～2.0%Triton-100, and (Dimethyl sulfoxid, DMSO), surplus be deionized water to 5.0%～15.0% dimethyl sulfoxide (DMSO).The screening formulation of solution B is: volume fraction is 1%Triton-100 and 10% dimethyl sulfoxide (DMSO), and surplus is deionized water.
3) the 150ml cell is separated spinning liquid, and its prescription can be: 0.2～0.5mol/LNaOH, 1～2mmol/LEDTA-Na
2, surplus is deionized water.Screening formulation is: 0.3mol/L NaOH and 1m mol/L EDTA-Na
2(pH=13.0), surplus is deionized water.
4) propidium iodide of DNA staining agent: 2.5ml 2 μ g/ml.
Embodiment 3
1) preparation of liver cancer SMMC7721 cell sample: liver cancer SMMC7721 cell cultures in the DMEM nutrient solution of 10% newborn calf serum, 100u/ml penicillin, 100u/ml Streptomycin sulphate, 37 ℃, CO
2Incubator is cultivated.Nutrient solution inclines when going down to posterity, wash twice with PBS, 0.25% pancreatin digests, the experimental degree of converging that cell inoculation need be cultivated 24h to 80% in 6 orifice plates, establish the dna double splitting of chain reagent etoposide effect 12h of 12.5 μ mol/L, 25 μ mol/L, 3 concentration of 50 μ mol/L then, other establishes the blank group.Finish action time, the PBS washed twice, the centrifugal 5min collecting cell of 1000r/m, stand-by with being diluted to cell suspension with the DMEM nutrient solution after the PBS washing 1 time again.
2) some glue: get liver cancer cell SMMC7721 cell suspension 20 μ l to be measured with behind 0.8% sepharose, the 60 μ l mixings of 37 ℃ of following preheatings, get in the gel slab sample well that 15 μ l point samples have made in the test kit of the present invention, each sample repeats more than 3.
3) lysis: before lysis, with an amount of solution A and solution B by being mixed and made into cell pyrolysis liquid at 1: 1, the gel slab that above-mentioned point sample is good immerses in the cell pyrolysis liquid of 4 ℃ of precoolings, embathes 2 times with the PBS lucifuge behind the lucifuge cracking 1h in 4 ℃ of environment, each 5min.
4) untwist and electrophoresis: will embathe the cell that good gel slab immerses 4 ℃ of precoolings and separate in the spinning liquid, in 4 ℃ of environment behind the lucifuge unwindase 13 0min, 150mA current stabilization electrophoresis 30min in 4 ℃ of environment.Electrophoretic buffer adopts the conventional TAE damping fluid in laboratory.
5) neutralization washing: the gel slab that will untwist embathes 2 times with 0.4mon/L Tris damping fluid (pH=7.5) lucifuge of 4 ℃ of precoolings, each 5min.
6) fluorescent dye: each sample well drips 10 μ l PI in the washed gel slab that will neutralize, and in 4 ℃ of environment behind the lucifuge dyeing 10min, washes unnecessary dye liquor with the 0.4mon/L Tris damping fluid (pH=7.5) of 4 ℃ of precoolings.
7) observation is taken pictures: the following 200 times of observations of fluorescent microscope, excitation wavelength 515～560nm.
The clear picture that the sample of present embodiment preparation shows has very high fluorescent brightness, and with background very high contrast gradient and resolving power (referring to Fig. 1) is arranged.Experimental result shows that all directed disengaging nucleus of the liver cancer SMMC7721 cell DNA fragment of damage forms typical comet image, and the hangover length and dna double splitting of chain reagent etoposide present dose-dependently, show that liver cancer SMMC7721 cell DNA degree of injury and etoposide have good dose-effect relationship.
Embodiment 4
1) preparation of liver cancer MHCC97-H cell sample: liver cancer MHCC97-H cell cultures is in the RPM1640 nutrient solution of 10% newborn calf serum, 100u/ml penicillin, 100u/ml Streptomycin sulphate, and 37 ℃, CO2 incubator are cultivated.Nutrient solution inclines when going down to posterity, wash twice with PBS, 0.25% pancreatin digests, the experimental degree of converging that cell inoculation need be cultivated 24h to 80% in 6 orifice plates, the etoposide effect 12h of 12.5 μ mon/L, 25 μ mon/L, three concentration of 50 μ mon/L then, other establishes the blank group.Finish action time, the PBS washed twice, the centrifugal 5min collecting cell of 1000r/m, stand-by with being diluted to cell suspension with the DMEM nutrient solution after the PBS washing 1 time again.
2) some glue: the suspension 20 μ l that get liver cancer MHCC97-H cell to be measured are with behind 1% sepharose mixing of the preheating of 37 ℃ of following preheatings, get in the gel slab sample well that 10 μ l point samples have made in the test kit of the present invention, and each sample repeats more than 3.
3) lysis: before lysis, with an amount of solution A and solution B by being mixed and made into cell pyrolysis liquid at 1: 1, the gel slab that above-mentioned point sample is good immerses in the cell pyrolysis liquid of 4 ℃ of precoolings, embathes 2 times with the PBS lucifuge behind the lucifuge cracking 1.5h in 4 ℃ of environment, each 5min.
4) untwist and electrophoresis: will embathe the cell that good gel slab immerses 4 ℃ of precoolings and separate in the spinning liquid, lucifuge is untwisted behind the 45min in 4 ℃ of environment, 150mA current stabilization electrophoresis 30min in 4 ℃ of environment.Electrophoretic buffer adopts the conventional TAE damping fluid in laboratory.
5) neutralization washing: the gel slab that will untwist embathes 2 times with 0.4mon/L Tris damping fluid (pH=7.5) lucifuge of 4 ℃ of precoolings, each 5min.
6) fluorescent dye: each sample well drips 10 μ l PI in the washed gel slab that will neutralize, and in 4 ℃ of environment behind the lucifuge dyeing 10min, washes unnecessary dye liquor with the 0.4mon/L Tris damping fluid (pH=7.5) of 4 ℃ of precoolings.
7) observation is taken pictures: the following 200 times of observations of fluorescent microscope, excitation wavelength 515～560nm.
The clear picture that the sample of present embodiment preparation shows has very high fluorescent brightness, and with background very high contrast gradient and resolving power (referring to Fig. 2) is arranged.Experimental result shows that all directed disengaging nucleus of the liver cancer MHCC97-H cell DNA fragment of damage forms typical comet image, and the hangover length and etoposide present dose-dependently, show that liver cancer MHCC97-H cell DNA degree of injury and etoposide have good dose-effect relationship.
Embodiment 5
1) preparation of liver cell Changliver sample: liver cell Changliver cultivates in the RPM1640 nutrient solution of 10% newborn calf serum, 100u/ml penicillin, 100u/ml Streptomycin sulphate, 37 ℃, the cultivation of CO2 incubator.The nutrient solution that inclines when going down to posterity is washed twice, 0.25% pancreatin with PBS and is digested, the experimental degree of converging that cell inoculation need be cultivated 24h to 80% in 6 orifice plates, and 10ng/ml, 100ng/ml neocarzinostatin effect 12h then, other establishes the blank group.Finish action time, the PBS washed twice, the centrifugal 5min collecting cell of 1000r/m, stand-by with being diluted to cell suspension with the DMEM nutrient solution after the PBS washing 1 time again.
2) some glue: the suspension 20 μ l that get liver cell Changliver to be measured are with behind 1.5% sepharose, the 60 μ l mixings of the preheating of 37 ℃ of following preheatings, get in the gel slab sample well that 25 μ l point samples have made in the test kit of the present invention, each sample repeats more than 3.
3) lysis: before lysis, with an amount of solution A and solution B by being mixed and made into cell pyrolysis liquid at 1: 1, the gel slab that above-mentioned point sample is good immerses in the cell pyrolysis liquid of 4 ℃ of precoolings, embathes 2 times with the PBS lucifuge behind the lucifuge cracking 1.5h in 4 ℃ of environment, each 5min.
4) untwist and electrophoresis: will embathe the cell that good gel slab immerses 4 ℃ of precoolings and separate in the spinning liquid, lucifuge is untwisted behind the 45min in 4 ℃ of environment, 150mA current stabilization electrophoresis 30min in 4 ℃ of environment.Electrophoretic buffer adopts the conventional TAE damping fluid in laboratory.
5) neutralization washing: the gel slab that will untwist embathes 2 times with 0.4mon/L Tris damping fluid (pH=7.5) lucifuge of 4 ℃ of precoolings, each 5min.
6) fluorescent dye: each sample well drips 10 μ l reagent IV in the washed gel slab that will neutralize, and in 4 ℃ of environment behind the lucifuge dyeing 10min, washes unnecessary dye liquor with the 0.4mon/L Tris damping fluid (pH=7.5) of 4 ℃ of precoolings.
7) observation is taken pictures: the following 200 times of observations of fluorescent microscope, excitation wavelength 515～560nm.
The clear picture that the sample of present embodiment preparation shows has very high fluorescent brightness, and with background very high contrast gradient and resolving power (referring to Fig. 3) is arranged.Experimental result shows that all directed disengaging nucleus of the liver cell ChangliverDNA fragment of damage forms typical comet image, and the hangover length and neocarzinostatin present dose-dependently, show that liver cell Changliver dna damage degree and neocarzinostatin have good dose-effect relationship.
Claims (10)
1. a cell DNA injury testing reagent box is characterized in that being provided with box body and reagent, and described reagent comprises that low melting-point agarose, cell pyrolysis liquid, cell separate spinning liquid and DNA staining agent;
Described cell pyrolysis liquid is made up of solution A and solution B, and the prescription of described solution A is: 2～3mol/LNaCl, 0.1～0.2mol/L EDTA-Na
2, 5～100mmol/L Tris, massfraction 0.5%～1.5% sodium sarcosinate, surplus is deionized water; The prescription of described solution B is that volume fraction is 1%～2%Triton-100,5%～15% dimethyl sulfoxide (DMSO), and surplus is deionized water, described solution A was mixed with solution B in 1: 1 by volume;
The prescription that described cell is separated spinning liquid is: 0.2～0.5mol/L NaOH, 1～2mmol/L EDTA-Na
2, surplus is deionized water;
Described DNA staining agent is propidium iodide, ethidium bromide, Hoechst 33342, hoechst 33358 or DAPI.
2. a kind of cell DNA injury testing reagent box as claimed in claim 1 is characterized in that the prescription of described solution A is: 2.5mol/L NaCl, 0.1mol/LEDTA-Na
2, 10mmol/LTris, massfraction are 1% sodium sarcosinate, surplus is deionized water; The prescription of described solution B is: volume fraction is 1%Triton-100 and 10% dimethyl sulfoxide (DMSO), and surplus is deionized water.
3. a kind of cell DNA injury testing reagent box as claimed in claim 1 is characterized in that described cell separates the prescription of spinning liquid and be: 0.3mol/L NaOH and 1mmol/L EDTA-Na
2, surplus is deionized water.
4. a kind of cell DNA injury testing reagent box as claimed in claim 1, the concentration that it is characterized in that described propidium iodide is 1～5 μ g/ml, the concentration of described ethidium bromide is 10～100 μ g/ml.
5. cell DNA damage detecting method is characterized in that may further comprise the steps:
1) cell DNA is damaged sample suspension and low melting-point agarose gel mixing gets mixed solution, again with the mixed solution point sample in the gel slab sample well;
2) gel slab behind the point sample is immersed lucifuge cracking in the cell pyrolysis liquid;
3) gel slab after the cracking is immersed cell and separate in the spinning liquid and untwist, the rear electrophoresis that finishes untwists;
4) the gel slab sample well behind the electrophoresis drips the dyeing of DNA staining agent, analyzing and testing result under fluorescent microscope.
6. a kind of cell DNA damage detecting method as claimed in claim 5, it is characterized in that in step 1), the massfraction of described low melting-point agarose gel is 0.8%～1.5%, and the volume ratio of described cell DNA damage sample suspension and low melting-point agarose gel is 1: 2～10; The volume of described point sample is 10～25 μ l/ sample wells; Described gel slab adopts the plate lid of 96 porocyte culture plates, and the depression that described plate covers is as sample well.
7. a kind of cell DNA damage detecting method as claimed in claim 6 is characterized in that described concave bottom is carved with cut.
8. a kind of cell DNA damage detecting method as claimed in claim 5 is characterized in that in step 2) in, described cell pyrolysis liquid carried out 4 ℃ of precoolings before using; Described cracked condition is: 4～10 ℃ of temperature, time 1～1.5h; After finishing, described cracking adopts PBS detergent gel plate 1～3 time, each 5～10min.
9. a kind of cell DNA damage detecting method as claimed in claim 5 is characterized in that in step 3), and described condition of untwisting is: 4～10 ℃ of temperature, time 30～45min; Described electrophoretic condition is: 4～10 ℃ of temperature, time 30～60min, electric current 50～150mA; After finishing, adopts described electrophoresis Tris damping fluid detergent gel plate 1～3 time, each 5～10min.
10. a kind of cell DNA damage detecting method as claimed in claim 5 is characterized in that in step 4), and described painted condition is: 4～10 ℃ of temperature, time 5～20min.
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| CN106568824A (en) * | 2016-11-08 | 2017-04-19 | 浙江大学 | Method for detecting DNA damage of mussels under stress of compound heavy metals |
| CN113029732A (en) * | 2021-03-09 | 2021-06-25 | 中南大学 | Operation device and operation method for single cell gel electrophoresis experiment |
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| CN104914237A (en) * | 2015-05-20 | 2015-09-16 | 常州大学 | Chemical waste water endocrine disruption genotoxicity test identification method |
| CN105543399A (en) * | 2016-02-26 | 2016-05-04 | 中国科学院水生生物研究所 | Method for detecting Microcystis cell DNA damage through fluorescence |
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| CN106018529A (en) * | 2016-07-07 | 2016-10-12 | 中国科学院重庆绿色智能技术研究院 | Kit using single cell gel electrophoresis for detecting cell DNA damage and method |
| CN106018529B (en) * | 2016-07-07 | 2019-03-12 | 中国科学院重庆绿色智能技术研究院 | A kind of method of single cell gel electrophoresis detection DNA Damage |
| CN106568824A (en) * | 2016-11-08 | 2017-04-19 | 浙江大学 | Method for detecting DNA damage of mussels under stress of compound heavy metals |
| CN113029732A (en) * | 2021-03-09 | 2021-06-25 | 中南大学 | Operation device and operation method for single cell gel electrophoresis experiment |
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