CN106556581A - Method for quick of the plasma to external DNA break damage strength - Google Patents
Method for quick of the plasma to external DNA break damage strength Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
Method for quick of the plasma to external DNA break damage strength, for determining the damage strength including the plasma method of mutagenesis including atmospheric pressure at room plasma (ARTP) to DNA, comprises the steps：1) DNA cladding microspheres are processed using plasma method of mutagenesis, and not plasma-treated matched group is set；2), after washing, DNA cladding microspheres are dyeed using dyestuff；3) fluorescent value that DNA after dyeing coats microsphere is determined using fluorescence detector；4) relative amount of DNA on microsphere after matched group and plasma mutagenic treatment is calculated according to fluorescent value, the gene damage intensity of plasma is judged.The present invention establishes a kind of method for quick of external DNA damage intensity using DNA cladding microspheres, so as to damage strength of the direct effective evaluation plasma to DNA.
Description
Technical field
The present invention relates to a kind of method for quick of plasma to external DNA break damage strength, specially utilizes
DNA cladding microspheres set up a kind of method for quick of external DNA damage intensity.
Background technology
Colory microbial strains are the core of biological industry all the time, how to be realized using efficient biological induced-mutation technique
The rapid Optimum of bacterial strain, is the important process of biological industry.
By physics (such as X-ray, gamma-rays, UV, ion beam mutation, plasma), chemistry (as alkylating agent,
Azide), the means such as biological (such as expression mutS, ndk gene) can be created more at short notice improving mutation rate
Many genotype.Different method of mutagenesis cause the mode of DNA damage different, as ultraviolet (UV) mainly causes Pyrimidine cyclobutane
Dimer (CPD) and 6-4 photoproducts (6-4PPs), so as to cause mutation (Sinha R P, the Hader D P.UV- of C to T
induced DNA damage and repair:a review.Photochem.Photobiol.Sci.,2002,1(4):225-236.).And
Chemical mutagen then mainly causes DNA alkylations to damage or generate base adduct, so as to cause base mispairing or DNA
Fracture (Miao Z-H, Rao V A, Agama K, et al.4-nitroquinoline-1-oxide induces the formation of
cellular topoisomerase I-DNA cleavage complexes.Cancer Research,2006,66(13):6540-6545.)。
Plasma technique is a kind of emerging mutagenic breeding new technique, can be in normal pressure, room using various plasma sources
Plasma, such as plasma torch, corona discharge plasma, dielectric barrier discharge plasma, radio frequency are produced under the conditions of temperature
Glow discharge plasma etc., wherein what is be applied to microorganism mutation and obtained good result is atmospheric pressure at room plasma
(Atmospheric and Room Temperature Plasma, ARTP), belongs to radio-frequency glow discharge plasma.
Wang Liyan utilizes Mass Spectra effects of the ARTP to synthetic oligonucleotide, finds ARTP direct effects
In oligonucleotide, the phosphodiester bond on oligonucleotide backbone, and the connection of base and pentose and amino are easily caused
Fracture (the Wang L Y.Studies on the mechanisms and applications of the atmospheric room of key
temperature plasmas acting on the microbes.Beijing:Department of Chemical Engineering,
2009.).O ' Connell et al. (O ' Connell D, Cox L J, Hyland W B, et al.Cold atmospheric
pressure plasma jet interactions with plasmid DNA.Applied Physics Letters,2011,98(4):043701.)
With Li et al. (Li G, Li H P, WangL Y, et al.Genetic effects of radio-frequency, atmospheric-pressure
glow discharges with helium.Applied Physics Letters,2008,92(221504):1-3.) with air jet flow cold etc.
Gas ions PBS solution respectively to plasmid and it is dried plasmid and processes, the plasmid after process is carried out into gel electrophoresiss and is tested
Card, it is found that ARTP can result in the open loop fracture of plasmid, plasmid be degraded into little DNA fragmentation.It can be seen that
ARTP is main injury pattern to the fracture damage of DNA.
Change the operating condition of ARTP mutagenesis systems, such as gas flow power illumination distance can change the operation of mutagenesis system
Condition, such as gas flow, power, irradiation distance can change the physical characteristics of ARTP, including active particle species, concentration
Deng (Li H P, Wang Z B, Ge N, et al.Studies on the physical characteristics of the radio-frequency
atmospheric-pressure glow discharge plasmas for the genome mutation of Methylosinus
Trichosporium.IEEE Transactions on Plasma Science,2012,40(11):2853-2860.).For ARTP is lured
The operating parameter of change system, generally makees parameter using power, generally using power (Pin) 20-120W, gas flow
(QHe) 5-20slpm, irradiation distance (D) are 2-5mm, but under the conditions of different operating, the dosage of ARTP does not have standard
Really determine.
This R＆D team qualitatively demonstrates fracture damages of the ARTP to DNA by the means of gel electrophoresiss, but mesh
Before till, lack the method for quick of DNA break damage strength, and then for characterizing the Dosages of ARTP.Comet
Method (single cell gel electrophoresis) be a kind of half-quantitative detection DNA break damage method (Kumari S, Rastogi R P,
Singh K L,et al.DNA damage:Detection strategies.Excli Journal,2008,7:44-62.), but its operation
Complexity, is only used for big DNA fragmentation.Therefore a kind of plasma including including ARTP of exploitation is needed badly to external DNA
The method for quick of fracture damage intensity.
The content of the invention
It is an object of the invention to provide a kind of method for quick of plasma to external DNA break damage strength, leads to
Measure DNA cladding fluorescent values of the microsphere before and after plasma mutation is crossed, the relative of DNA on microsphere is calculated before and after mutation
Amount, so as to detect the gene damage intensity of plasma.
DNA break damage strength is proportional to the cleavage points of DNA, the DNA fragmentation of different length after to fracture
Separated, and it is quantitative respectively, DNA break number of sites can be calculated, so as to obtain DNA break damage strength.So
And, plasma is random fracture for DNA fragmentation, and the DNA fragmentation after being processed is degraded to the little of different length
DNA fragmentation, therefore be difficult the little DNA fragmentation of all different lengths after corona treatment is separated.
According to DNA claddings microsphere (synthetic method is shown in Fig. 2) of autonomous synthesis, the length of DNA is in tens bp thereon
The microsphere of double-stranded DNA, preferred length 100bp or so are coated with to before the sequencing of Roche 454 between thousand of bp, may also be employed
DNA be connected on microsphere.Plasma is cut off at random to the DNA on microsphere, the Dosages and microsphere of plasma
The amount of damage of upper DNA is proportionate.Detected at not plasma-treated matched group and Jing plasmas by fluorescence staining
The fluorescent value of microsphere DNA in the experimental group of reason, on the basis of DNA relative amounts on matched group microsphere, you can obtain DNA
Fracture damage intensity.
Therefore, technical scheme is as follows：
Method for quick of the plasma to external DNA break damage strength, it is characterised in that comprise the steps：
1) DNA cladding microspheres are processed using plasma method of mutagenesis, and not plasma-treated matched group is set；
2) the DNA cladding microspheres and matched group of the process of Jing plasmas method of mutagenesis are washed respectively, and is distinguished using dyestuff
Dyeing；
3) fluorescence that the DNA that Jing plasmas method of mutagenesis is processed after dyeing coats microsphere and matched group is determined using fluorescence detector
Value；
4) according to the fluorescent value that 3) determines calculate matched group and Jing after plasma mutagenic treatment on microsphere DNA relative quantity, judge
The gene damage intensity of plasma.
The DNA claddings microsphere is that DNA coats agarose microbeads, and each DNA cladding agarose microbeads can connect 10-
The DNA of 10000bp, the DNA of preferred 100bp.
Step 1) in, DNA claddings microsphere is resuspended using deionized water afterwards before any plasma treatment, is utilizing plasma
Guarantee that the deionized water on microsphere has been evaporated completely during body process, specifically DNA cladding microspheres can be spread evenly across glass dish
In, it is placed in super-clean bench to deionized water and evaporates complete, then the mutagenic treatment for carrying out plasma.
Step 2) in, the dyestuff is nucleotide dye, specifically optional molecular probe SYBR Green I nucleotide dye
Material.The dyestuff is to detect one of most sensitive dyestuff of double chain DNA molecule, the amplified production being normally used in quantitative PCR.
SYBR Green I fluorescent values itself are relatively low, and more than 1000 times of rear Fluorescence Increasing, therefore conduct are being combined with DNA molecular
Its background value of DNA dyestuffs is low.The conventional nucleic acid dye ethidium bromide of the remolding sensitivity of SYBR Green I is high more than 50 times, can
The DNA of minimum 20pg is taken to detect agarose or polyacrylamide gel electrophoresis bar.
Step 2), before dyeing, using deionized water fully wash DNA cladding microsphere, by degraded DNA fragmentation with
DNA microspheres after process are separated.The dyeing, is that DNA is coated the TAE bufferings that microsphere is resuspended in SYBR Green I
In liquid, and in the static dyeing 1h in room temperature dark place.In theory, the Sybr Green I dyes of a molecule are may be inserted in every 3bpDNA
Material.DNA of the lowest detection lower limit of SYBR Green I nucleotide dyes for 20pg.
Step 3) in, the fluorescence detector is multi-functional micropore board detector, can select many work(of Infinite 200PRO
Energy micropore board detector (Tecan,Switzerland fluoroscopic examination) is carried out, excitation wavelength is 497nm, is launched
Wavelength is 520nm, and detection Z axis distance and yield value adopt Automatic Optimal result.
In said method, the plasma is atmospheric pressure at room plasma, and the atmospheric pressure at room plasma is by exposed gold
Category electrode is produced under atmospheric pressure, room temperature condition.
In said method, the plasma can also be corona discharge plasma, dielectric barrier discharge plasma or
Other radio-frequency glow discharge plasmas outside atmospheric pressure at room plasma.
Beneficial effect：Method for quick of the plasma that the present invention is provided to external DNA break damage strength, need not
Accurately know total microsphere number and DNA content, need to only pass through to determine the fluorescent value of DNA on microsphere, obtain ARTP process
In front and back on microsphere DNA relative quantity, so as to characterize damage strength of the plasma jet to DNA under the conditions of different operating,
Therefore the method is quick, easy；Meanwhile, compared to comet method, (single cell gel electrophoresis, a kind of half-quantitative detection DNA break
The method of rhegma wound) --- big DNA fragmentation is only used for, the method is suitable for the tens DNA pieces arrived between thousand of bp
Section, the scope of application are wider, and the application of plasma mutagenesis system and the research of mutagenic character and mechanism are significant.
Description of the drawings
Fig. 1 is to determine flow charts of the ARTP to external DNA break damage strength.
Fig. 2 is the building-up process schematic diagram that DNA coats microsphere.
Fig. 3 is the micro- photograph that ARTP before processings (a, b) and ARTP processes that 120s (c, d) DNA coats microsphere
Piece, ARTP mutagenic conditions are QHe=10slpm, D=2mm, Pin=100W, t=120s.
Graphs of a relation of the Fig. 4 for fluorescent value at the DNA relative concentrations and 520nm on DNA cladding microspheres.
Fig. 5 is the pass that different ARTP process times and DNA coat microsphere 520nm fluorescent values and relative dna content
System's figure, ARTP mutagenic conditions are QHe=10slpm, D=2mm, Pin=100W.
Fig. 6 is different ARTP gas flows and DNA coats the graph of a relation of microsphere 520nm fluorescent values, ARTP mutation bars
Part is D=2mm, Pin=100W, t=45s.
Fig. 7 is different ARTP outputs and DNA coats the graph of a relation of microsphere 520nm fluorescent values, ARTP mutation bars
Part is QHe=10slpm, D=2mm, t=90s.
Fig. 8 is different ARTP stream distances and DNA coats the graph of a relation of microsphere 520nm fluorescent values, ARTP mutation bars
Part is QHe=10slpm, Pin=100W, t=45s.
Wherein, ARTP- atmospheric pressure at room plasma, QHe- gas flow, D- samples and plasma jet entrance away from
From Pin- input power, t- process times.
Specific embodiment
With in plasma atmospheric pressure at room plasma (Atmospheric and Room Temperature Plasma,
ARTP, as a example by), the method for the present invention is illustrated.
Fig. 1 is to determine schematic diagrams of the ARTP to external DNA break damage strength.DNA is processed first with ARTP
Cladding microsphere, the active particle that ARTP is produced pass through reaction or energy transmission with the DNA on microsphere, at random on microsphere
DNA is ruptured.There is from after different loci fracture the DNA fragmentation of different length, depart from from microsphere, be free in micro-
In aqueous solution outside ball.Just the DNA fragmentation of degraded can be divided with the DNA microspheres after process by the method for centrifuge washing
From.Using SYBR Green to the DNA cladding microsphere dyeing after process, can just determine micro- using fluorescence spectrophotometry
The amount of surplus DNA on ball.Contrasted with the amount of DNA on before processing microsphere, can just be obtained DNA after certain process time
The ratio of fracture damage, so as to realize measure of the ARTP to DNA break damage strength.
1st, DNA cladding agarose microbeads are prepared
Using recovery microsphere of the DNA cladding microspheres before the sequencing of Roche 454, carried by Hua Da gene (Shenzhen, China)
For.
Fig. 2 gives the building-up process of 454 sequencing microspheres：Covalently tie on the magnetic microsphere of 1 microns of diameter first
Streptavidin is closed, these microspheres is cultivated 30 minutes under room temperature in the buffer containing oligonucleotide, in oligonucleotides
5 ' ends of acid are modified by double biotin groups, therefore these oligonucleotides can be adsorbed onto on microsphere with affine.Early stage text
Offer report (Dressman D, Yan H, Traverso G, et al.Transforming single DNA molecules into
fluorescent magnetic particles for detection and enumeration of genetic variations.Proceedings of
the National Academy of Sciences of the United States of America,2003,100(15):8817-8822.),
Probably absorption 10 on each microsphere5Individual oligonucleotide fragment.These microspheres with oligonucleotide, in 454 sequencing procedures
In, it is connected with the DNA fragmentation of about 100bp or so to be sequenced by emulsion-based PCR, and amplification forms the bag of DNA
Cover.Unloaded microsphere is removed by selected by flow cytometry apoptosis, you can for the external DNA damage intensity detection experiment of ARTP
Explore.
2nd, using the change of microsphere after micro- sem observation ARTP before processings
To check that ARTP processes the degree of impairment to microsphere itself, using Nicon ECLIPSE Ti (Tokyo, Japan) microscopes
The metamorphosis of microsphere after observation ARTP before processings.
If microsphere can be degraded by the active particle that ARTP is produced, then in follow-up washing process, a part
DNA molecular can be washed off so as to bring the loss of DNA with broken microsphere.In order to detect ARTP for the effect of microsphere,
Gas flow 10slpm, irradiation distance 2mm, under conditions of input power 100W, microexamination ARTP irradiation 120s
In front and back, DNA coats the metamorphosis of microsphere.
From Fig. 3, the result of microexamination is found out, when ARTP processes 120s, the plasma that ARTP is produced will not
The broken of microsphere itself is caused, therefore the loss of DNA will not be caused and caused error because microsphere is broken.
3rd, DNA cladding agarose microbeads are processed using ARTP
ARTP (atmospheric pressure at room plasma) mutagenic breeding instrument, model ARTP- II are raw by this R＆D team and Wuxi source clear sky wood
Thing Science and Technology Ltd. researches and develops (can buy from market) jointly.
DNA cladding microspheres are dissolved in 1 × TAE buffer, 5,000 × g is centrifuged 2min under room temperature, remove supernatant,
With the deionized water of equivalent it is resuspended after, 5,000 × g is centrifuged 2min under room temperature, so in triplicate, with remove DNA cladding
The DNA fragmentation dissociated in microspheres solution, prevents from bringing error to subsequent experimental.
The aqueous solution that microsphere is coated containing DNA after process, after concussion is mixed, draws 20 μ l and uniformly coats diameter
In the lucite plate of 16mm, place in super-clean bench, be used for the process of follow-up ARTP to moisture evaporation completely afterwards.
The ARTP working gas adopted in the present embodiment is helium (He) of the purity more than 99.995%, adopts in embodiment
Distance, the irradiation time of gas flow, input power, sample and plasma jet entrance, as needed in certain model
It is adjusted in enclosing.Microsphere after ARTP process, it is fully resuspended with 200 μ l deionized waters, collect 1.5ml EP pipes
In, and be washed with deionized 3 times, determine for follow-up DNA dyeing.
4th, dyeed to coating DNA on microsphere using nucleotide dye
4.1 select SYBR Green nucleotide dyes
Molecular probe SYBR Green nucleotide dyes are one of most sensitive dyestuffs of detection double chain DNA molecule, and it is fixed to be normally used for
In amount PCR, amplified production is quantitative.SYBR Green fluorescent values itself are relatively low, and rear Fluorescence Increasing is being combined with DNA molecular
More than 1,000 times, therefore its background value is low as DNA dyestuffs.The conventional nucleic acid dye bromine of the remolding sensitivity of SYBR Green
Change second ingot high more than 50 times, agarose can be detected or polyacrylamide gel electrophoresis bar takes the DNA of minimum 20pg.
DNA cladding microspheres solutions are dyeed using SYBR Green in the present embodiment, SYBR Green are 10000
×, using after 10000 times of dilution, concrete concentration may refer to Sigma websites.Due to connecting oligonucleotide on each microsphere
The number of joint is about 105It is individual, and the DNA fragmentation length on microsphere after PCR amplifications is about 100bp.Thus calculate,
Assume that each oligonucleotide adapters connects a DNA fragmentation, then the DNA content on each microsphere is about 0.01pg.
The Sybr Green dyestuffs of a molecule are may be inserted in per 3bpDNA, the Monitoring lower-cut of SYBR Green is the DNA of 20pg
(Karlsen F,Steen H B,Nesland J M.SYBR Green I DNA staining increases the detection
sensitivity of viruses by polymerase chain reaction.Journal of Virological Methods,1995,
55(1):153-156.), then the DNA content on 2,000 microsphere can be at least detected in theory.
4.2 are dyeed using SYBR Green nucleotide dyes
SYBR Green I nucleotide dyes are purchased from SigmaAldrich companies (St.Louis, MO, USA).10,000 × SYBR
The DMSO solution of Green I is in -20 DEG C of preservations.It is diluted using 1 × TAE (pH 8.0) buffer is front used.To process
And the DNA microspheres after washing are resuspended in the TAE solution of SYBR Green I of 200 μ l, room temperature dark place is static to dye 1
h。
4.3 fluorescent values for determining microsphere DNA
DNA microsphere suspension liquids after SYBR Green dyeing, using the multi-functional micropore board detectors of Infinite 200PRO
(Tecan,Switzerland fluoroscopic examination) is carried out, excitation wavelength is 497nm, and launch wavelength is 520nm,
Detection Z axis distance and yield value adopt Automatic Optimal result.
4.4SYBR Green dye setting-out line scope
Coat microsphere to determine breakdown rates of the ARTP to DNA double chain using DNA in the present embodiment, without the need for accurately knowing total
Microsphere number and DNA content, it is only necessary to know the relative quantity of DNA on microsphere after ARTP before processings.Therefore determine micro-
Under dilution variable concentrations, SYBR Green dye the quantitative range of linearity to ball.As a result as shown in fig. 4 a.Microsphere is carried out by
Level dilution, each extension rate are 5.When dilution 4 times, microsphere concentration for original concentration 1.6 × 10-3When, using SYBR
Green dyeing is determined still in linearly interval, illustrates SYBR Green dyeing setting-out line wide ranges.
And ARTP is irradiated to microsphere, maximum damage strength is 50% of DNA content on microsphere.This be due to wait from
Daughter cannot be applied to the DNA in microsphere the latter half, and this part DNA cannot occur fracture.Therefore determine from dilution
0.2 times of SYBR Green dyeing quantitative linearity curve to original microsphere, as shown in Figure 4 b.
Linear fit is carried out to the result that Fig. 4 b are obtained, following fitting result is obtained：
Y=44179x,
R2=0.98.
From result above, the slope that SYBR Green dyeing is determined is 44179, shows that the sensitivity for determining is high.Say
Bright SYBR Green can be used in the accurate quantitative analysis of follow-up DNA microspheres.
Fracture damage intensity of the DNA under different plasma treatment conditions on 5 analysis microspheres
According to DNA relative amounts on microsphere and the relation of fluorescent value, with the DNA content on not plasma-treated microsphere it is
Benchmark, is set to matched group, that is, determine its fluorescent value, and its DNA relative amount is set as 1, and pass through to determine plasma
After body process on microsphere DNA fluorescent value, with reference to the graph of a relation of DNA relative amounts-fluorescent value, after being processed on microsphere
The relative amount of DNA, is finally compared with DNA relative amounts on undressed microsphere, is obtained DNA on microsphere and is existed
Fracture damage intensity under different plasma treatment conditions.
The DNA of fracture damage intensity=(the DNA relative amounts of the DNA relative amounts-treatment group of matched group)/matched group
Relative amount
6th, fracture damage intensity of the ARTP different disposals condition to external DNA
6.1ARTP processes impact of the different time to external DNA damage intensity
Fixed gas flow is 10slpm, and input power is 100W, and sample is 2mm with the distance of plasma jet entrance, is adjusted
Section process time is respectively 0s to 120s, determines ARTP and processes fracture damage intensity of the different time to DNA.
As SYBR Green dyeing background values are low, fluorescence intensity is directly proportional to relative dna content.As a result such as Fig. 5 institutes
Show, wherein Fig. 5 a are the graph of a relation that ARTP process times and DNA coat microsphere fluorescence value, when Fig. 5 b are that ARTP is processed
Between with corresponding DNA coat microsphere on relative dna content graph of a relation.At the initial stage of process, the DNA double chain on microsphere contains
Amount is higher, and with the growth of ARTP process times, the fluorescent value of microsphere is reduced rapidly, when treated between when reaching 60s, with
The continuation of ARTP process times increases, and the DNA content change on microsphere is little.This stop mainly due to microsphere itself
Effect, causes in microsphere the latter half, i.e., dorsad the half DNA double chain of ARTP jet directions cannot be produced with ARTP
Active particle effect, therefore the maximum DNA loss amount on microsphere be 50%.
Impact of the 6.2ARTP gas flows to external DNA damage intensity
Fixed plasma input power 100W, sample and plasma jet entrance distance are 2mm, and irradiation time is 45s, gas
Body flow is adjusted in the range of 3slpm to 15slpm, determines fracture damage intensity of the ARTP to DNA microspheres.
As a result as shown in fig. 6, being, in the range of 0slpm to 9slpm, to increase with gas flow in gas, DNA break is damaged
Hinder intensity increase, illustrate to fix other operating parameters, increase with gas flow, ARTP acts on the dosage increase of DNA.
When gas flow continues to increase to 15slpm more than 9slpm, increase with gas flow, the fracture damage intensity of DNA is no longer
Increase.Illustrate under this operating condition, gas flow is higher than 9slpm, continues the Dosages shadow of increase gas flow ARTP
Ring little.
Impact of the 6.3ARTP input powers to external DNA damage intensity
Fixed sample and plasma jet entrance distance are 2mm, and gas flow is 10slpm, and irradiation time is 90s, plasma
Input power is adjusted in the range of 20W to 120W, determines fracture damage intensity of the ARTP to DNA.
As a result as shown in fig. 7, input power be less than 90W when, with the increase of input power, DNA break damage strength
Strengthen, be higher than 90W in input power, continue increase input power and DNA break damage strength is not significantly increased.Explanation
In certain input voltage range, increase the ionization degree that voltage can improve two interpolar gas of plasma generator, cause
ARTP Dosages are raised, and after input power increases to certain value, are raised with voltage, and fluerics gas temperature is raised, and is gone
Degree of ionization increases, therefore the Dosages of ARTP do not continue to increase.
Impact of the 6.4ARTP irradiation distances to external DNA damage intensity
Fixed ARTP input powers are 100W, and gas flow is 10slpm, and irradiation time is 45s, changes sample and plasma
The distance of jet inlet, determines its impact to DNA break damage strength.
As a result as shown in Figure 8 a, in the range of plasma jet entrance 6mm, increase with distance, DNA's is disconnected
Split damage strength not to be reduced.The result can not illustrate ARTP produce plasma with stream distance increase without become
Change.With the increase of stream distance, the active particle in plasma is constantly collided with the gas molecule in air, by energy
Amount and momentum are transferred to gas molecule, generate some new active substances so as to react.In stream distance 6mm, due to sky
Gas convection current is not obvious, and the energy dissipation caused by particle encounter is not obvious, and the active particle in plasma is produced with reaction
The energy that new active substance has is still very high, and DNA double chain can be ruptured.When stream distance continues to increase,
When reaching 8mm, as convection current influence is strengthened, cause energy dissipation substantially, to the fracture damage intensity of DNA
Reduce.
Continue to increase distance of the sample in jet downstream, determine the fracture damage intensity of DNA, as a result see Fig. 8 b.It was found that
The fracture damage intensity of DNA is substantially reduced, and is illustrated when distance increases to 10mm, by atmospheric environment convection current and plasma
Energy dissipation impact, the Dosages of ARTP are reduced rapidly, cause the damage strength to DNA to reduce.Result above
Illustrate, when being processed to biological sample using ARTP, the distance of sample and jet inlet within 6mm,
Beyond the scope, ARTP will be reduced to the treatment effeciency of sample.
Claims (10)
1. method for quick of the plasma to external DNA break damage strength, it is characterised in that comprise the steps：
1) DNA cladding microspheres are processed using plasma method of mutagenesis, and not plasma-treated matched group is set；
2) the DNA cladding microspheres and matched group of the process of Jing plasmas method of mutagenesis are washed respectively, and is distinguished using dyestuff
Dyeing；
3) fluorescence that the DNA that Jing plasmas method of mutagenesis is processed after dyeing coats microsphere and matched group is determined using fluorescence detector
Value；
4) according to the fluorescent value that 3) determines calculate matched group and Jing after plasma mutagenic treatment on microsphere DNA relative quantity, judge
The gene damage intensity of plasma.
2. the method for claim 1, it is characterised in that the DNA claddings microsphere is that DNA coats agarose microbeads,
Each DNA cladding agarose microbeads can connect the DNA of 10-10000bp.
3. method as claimed in claim 2, it is characterised in that the DNA claddings agarose microbeads can connect 100bp's
DNA。
4. the method for claim 1, it is characterised in that step 2) dyestuff is nucleotide dye.
5. method as claimed in claim 4, it is characterised in that the nucleotide dye is SYBR Green I nucleotide dyes,
The dyeing, in the static dyeing 1h in room temperature dark place.
6. method as claimed in claim 5, it is characterised in that the lowest detection lower limit of the SYBR Green I nucleotide dyes
For the DNA of 20pg.
7. the method for claim 1, it is characterised in that step 3) fluorescence detector is the detection of multi-functional microwell plate
Instrument.
8. method as claimed in claim 7, it is characterised in that the excitation wavelength of the multi-functional micropore board detector is
497nm, launch wavelength are 520nm.
9. the method as described in claim 1-8 is arbitrary, it is characterised in that the plasma is atmospheric pressure at room plasma, described
Atmospheric pressure at room plasma is produced under atmospheric pressure, room temperature condition by bare metal electrode.
10. the method as described in claim 1-8 is arbitrary, it is characterised in that the plasma include corona discharge plasma,
Dielectric barrier discharge plasma and radio-frequency glow discharge plasma.
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