CN1945332A - Method for detecting pyrimidine dipolymer content on DNA chain - Google Patents
Method for detecting pyrimidine dipolymer content on DNA chain Download PDFInfo
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- CN1945332A CN1945332A CN 200610096266 CN200610096266A CN1945332A CN 1945332 A CN1945332 A CN 1945332A CN 200610096266 CN200610096266 CN 200610096266 CN 200610096266 A CN200610096266 A CN 200610096266A CN 1945332 A CN1945332 A CN 1945332A
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- pyrimidine
- dipolymer
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- pyrimidine dimer
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Abstract
The process of detecting pyrimidine dipolymer content on DNA chain includes the following steps: fixing one DNA probe with biotin modified end and one pyrimidine dipolymer on the surface of chip; preparing standard pyrimidine dipolymer; mixing standard pyrimidine dipolymer in 0-80 nM and pyrimidine dipolymer antibody in 0.2-5 mcg/ml, and mapping the standard curve of response value of detecting surface plasma resonant sensor mixture liquid flowing through the chip surface vs the concentration of pyrimidine dipolymer; mixing the measured sample with antibody in the same concentration as that in establishing standard curve and detecting the response value of the mixed liquid flowing through the chip surface so as to obtain the concentration of pyrimidine dipolymer in the sample based on the standard curve. The present invention has high detection speed, high accuracy, high repeatability and other advantages and may be used widely.
Description
Technical field:
The invention belongs to the detection method technical field of gene mutation, particularly the assay method of pyrimidine dipolymer content and the immunologic detection method of sudden change on the DNA chain.
Background technology:
According to " fragrance flavor and cosmetic " (2005.6:25-28,32) report, ultraviolet irradiation can cause dna damage, forms pyrimidine dimer (CPD).The formation of pyrimidine dimer can cause various disease conditions such as scytitis, skin senescence even cutaneum carcinoma.The content of pyrimidine dimer has reflected the DNA extent of damage, can be used for validity of estimating various preventions and treatment means etc.The quantitative measurement pyrimidine dipolymer content is the necessary means of research ultraviolet damage, and therefore, people are exploring effective detection method always.
The common method of measuring CPD concentration at present comprises: Germany " MGG " (Mol Gen Genet, 1982,186.4:475-7) radioimmunology (RIA) mentioned, U.S.'s " analytical biochemistry " (Anal Biochem, 2004,329.2:263-8) high performance liquid chromatography (HPLC) used, Holland " environmental science " (J Environ Sci, 2004,16.1:173-6) euzymelinked immunosorbent assay (ELISA) (ELISA) and U.S.'s " photochemistry and photobiology " (the Photochem Photobiol that use, 1996, the gas chromatography mass spectrometry method of 64.2:310-5) mentioning (GC-MS).Wherein radioimmunology is eliminated gradually owing to will use isotope.High performance liquid chromatography can only be analyzed the substrate of length unanimity, can't be used for detecting intracellular pyrimidine dipolymer content.The gas chromatography mass spectrometry method can only detect more single substrate owing to specimen preparation in the whole process is loaded down with trivial details, to having relatively high expectations of tester, and needs two high-accuracy instruments, and is therefore also difficult to promote the use of.ELISA is a method easy relatively, that practicality is wider, the ELISA methods that adopt in the article of delivering in recent years more.But because the ELISA method needs the plate of washing of envelope antigen and repeated multiple times, also need two anti-marks and colour developing etc., step is many, complex operation, and required time is longer; Because operation steps is many, be easy to generate bigger operate miss simultaneously, thereby cause shortcomings such as quantitatively not accurate enough.Therefore the technology of developing fast quantification detection CPD has important and practical meanings.
Summary of the invention:
The objective of the invention is to propose a kind of method of utilizing immunity competition principle to measure pyrimidine dipolymer content on the DNA chain, but to overcome shortcomings such as existing method complex operation, sample preparation complexity test sample narrow range even contaminated environment.
The present invention measures the method for pyrimidine dipolymer content on the DNA chain, it is characterized in that: synthetic one section only contains the dna probe that two pyrimidine bases are in the adjacent position earlier, and it is terminal through biotin modification; After ultraviolet irradiation makes two adjacent pyrimidine bases form pyrimidine dimer, this dna probe is fixed to chip surface; Synthetic again one section end is not with modification and only contain the single stranded DNA that two pyrimidine bases are in the adjacent position, with ultraviolet ray irradiation adjacent pyrimidine bases are formed after the pyrimidine dimer, separate the standard items that obtain pyrimidine dimer through reversed-phase high-performance liquid chromatography (RP-HPLC); Getting the pyrimidine dimer standard items of variable concentrations more than five kinds that comprise the border between 0～80nM mixes with the pyrimidine dimer antibody of specific concentrations in 0.2～5 μ g/ml scope respectively, detect the response that produces when mixed liquor flows through chip surface by surface plasma resonance sensor, response to the mapping of pyrimidine dimer concentration, is obtained the typical curve of pyrimidine dimer; Testing sample is mixed with the antibody of setting up the typical curve same concentrations, detect mixed liquor and flow through the response that chip produces, itself and typical curve are contrasted, can draw the concentration of pyrimidine dimer in the sample.
If the length of DNA chain surpasses 500bp in the testing sample, then need it is interrupted, form the following short chain of 500pb, measure again.
The present invention has utilized the principle of immune competition, the dna probe that will contain pyrimidine dimer is coupled to chip surface, the testing sample that contains pyrimidine dimer mixes with the antibody of specific recognition pyrimidine dimer, detects the response that produces when mixed liquor flows through chip surface by surface plasma resonance sensor.
Because a part of antibody CPD preferential and in the sample combines, so after antibody and the sample mix, the antibody amount that can cause being attached to chip surface is compared to some extent when flowing through antibody separately and is reduced, and promptly knows the concentration of pyrimidine dimer in the testing sample according to the reduction of antibody.The present invention and Holland's " environmental science " (J Environ Sci, 2004,16.1:173-6) euzymelinked immunosorbent assay (ELISA) of Shi Yonging is compared, and its advantage has: 1. detect fast, whole process can be finished in a few minutes; 2. quantitatively accurately, favorable reproducibility; 3. chip used can the repeated use, it is low to detect cost; 4. detectable sample scope is wide.The inventive method can be widely used for detection, research ultraviolet damage, the dna damage reparation to DNA of light repairase activity, and estimates depletion of the ozone layer to numerous areas such as health effects.
Description of drawings
Embodiment
The mensuration of embodiment 1, pyrimidine dimer (CPD) concentration
1. prepare the CPD standard items
The preparation of pyrimidine dimer study
Employing is given birth to synthetic one section of worker company by Shanghai and is only contained the oligonucleotide chain that two pyrimidine bases are in the adjacent position, have only two pyrimidine bases to be in the adjacent position in the chain, purpose is to guarantee only can form a pyrimidine dimer on every chain, is convenient to pyrimidine dimer is carried out accurately quantitatively.The oligonucleotides chain-ordering that uses in the present embodiment is 5 '-AGA GCA GTT GACACG-3 '.The 150 μ M oligonucleotide aqueous sample that 100 μ l contained 20% acetone are positioned over and can not inflate bubbling 15 minutes with the high-purity argon gas of purity＞99.999% through in the glass core magnetic tube less than the 290nm wavelength light, seal with sealing film.Ready sample is 25 ℃ with 300 watts of high voltage mercury lamp radiations, bath temperature, and the lamp distance is 5cm.The sample of irradiation after 1 hour is taken out in the centrifuge tube of 1.5ml, with 13,000 rev/mins speed centrifugal 10 minutes, gets supernatant with anti-phase high pressure liquid chromatography (RP-HPLC) detection or purifying, or be positioned in-20 ℃ of refrigerators standby.
The reversed-phase high-performance liquid chromatography of CPD oligonucleotide (RP-HPLC) detects and purifying
The condition that adopts is:
Mobile phase A: 7% acetonitrile (ACN), 0.1M triethylamine acetic acid esters (TEAA), pH 7.0
Mobile phase B: 10%ACN, 0.1M TEAA, pH 7.0
Pump type: Waters 600
Detecting device: Waters 2487 ultraviolet double-channel detectors
Chromatographic column: Gemini C18 column (50 * 4.60mm, 110 , F door)
Elution program: 0-5min, 7%ACN and 100mM TEAA (pH7.0): 5-35min are 7-10%ACN and 100mM TEAA (pH 7.0)
Flow velocity: 0.75ml/min
Column temperature: 60 ℃
Detection mode: 260nm uv absorption
Applied sample amount: 20 μ l
Get sample after the above-mentioned irradiation of 20 μ l with micro syringe, be injected to reversed-phase column, carry out RP-HPLC and analyze, collect the sample of 10.8 minutes chromatographic peaks, and analyze and quantitative, obtain the CPD standard items with Millennium 32 softwares of Waters.
2. dna probe is coupled to chip surface
1) will synthesize one section only contains the 5 ' end that two pyrimidine bases are in the dna probe of adjacent position and adds biotin modification (have only two pyrimidine bases to be in the adjacent position in the chain, purpose is to guarantee only can form a pyrimidine dimer on every chain).
The dna probe sequence of using in the present embodiment is:
Biotin-(CH
2)
6-AGA GCA GTT GAC ACG-3 ';
Standby by being stored in-20 ℃ after the top condition irradiation;
2) the SA chip is installed on the BIACORE3000 instrument, the method that the coupling connection of instrumentation and probe all provides by BIACORE company; Wash chip surface three times with 1M NaCl/50mM NaOH, each one minute; The CPD probe that shone is diluted to 1 μ M with the PBS damping fluid,, thereby CPD is coupled on the chip surface by sample on the 5 μ l/min flow velocitys 10 minutes.
Described dna probe is end modified, except the used biotin modification of present embodiment, can also be with amido modified, and the mode that joins by the covalency coupling is connected to chip surface with probe; Also can use sulfydryl modification, directly dna probe is assembled on the chip surface.
3. the foundation of typical curve
1), the preparation of sample will be bought the antibody (production code member: T1192) with 1000 times of the PBS dilutions that contains the 1mg/ml bovine serum albumin(BSA) in SIGMA company, the antibody of dilution is distributed into every pipe 150ul, the CPD standard items that prepare above are diluted to 0,1.25,2.5,5,10,20,40,80 and 160nM with PBS, respectively get 150ul and join in the antibody; Make that the antibody final concentration is 1 μ g/ml in the mixed liquor, the concentration of CPD is respectively 0,0.625,1.25,2.5,5,10,20,40,80nM.(annotate: the variation meeting of antibody concentration exerts an influence to the sensing range of this method, and the low concentration antibody of 0.2ug/ml can be relatively more responsive to the CPD of low concentration, but the range of linearity is narrow; The high concentration antibody of 5ug/ml can be measured denseer sample, and the range of linearity is wide, but insensitive to the CPD of low concentration.Taken all factors into consideration sensitivity and linear detection range, the used antibody concentration of present embodiment is 1mg/ml).
2), test is made as 5 μ l/min with surface plasma resonance sensor BIACORE3000 system flow rate, the compound sample 15 μ l of above-mentioned antibody of last sample and CPD, collection is in conjunction with the signal of peak; Then flow velocity is made as 30 μ l/min, regenerated in 30 seconds, wait for 2min with the 10mMNaOH sample introduction; Carry out the next round analysis again, each concentration is triplicate at least.
The result who tests above maps with origin software, horizontal ordinate is represented the concentration of pyrimidine dimer, ordinate is represented the response that records, the typical curve that obtains as shown in Figure 1: horizontal ordinate is represented the concentration of pyrimidine dimer among the figure, and ordinate is represented measured response.
4. the test of sample
Sample is mixed with the antibody of setting up the typical curve same concentrations, mixed liquor is flow through chip surface and detects response, compare, can obtain the concentration of pyrimidine dimer in the unknown sample with typical curve.If sample is plasmid or long-chain DNA, for example detect the content of CPD on the interior genomic DNA of cell, sample must be interrupted, can handle with the method for ultrasonic or high speed shear, form the following short chain of 500pb, otherwise can influence the accuracy of measurement result.
Claims (2)
1. a method of measuring pyrimidine dipolymer content on the DNA chain is characterized in that: synthesize one section earlier and only contain the dna probe that two pyrimidine bases are in the adjacent position, and it is terminal through biotin modification; After ultraviolet irradiation makes two adjacent pyrimidine bases form pyrimidine dimer, this dna probe is fixed to chip surface; Synthetic again one section end is with modifying and only contain the single stranded DNA that two pyrimidine bases are in the adjacent position, with the ultraviolet ray irradiation adjacent pyrimidine bases formed after the pyrimidine dimer, obtains the standard items of pyrimidine dimer through the reversed-phase high-performance liquid chromatography separation; Getting the pyrimidine dimer standard items of variable concentrations more than five kinds that comprise the border between 0～80nM mixes with the pyrimidine dimer antibody of specific concentrations in 0.2～5 μ g/ml scope respectively, detect the response that produces when mixed liquor flows through chip surface by surface plasma resonance sensor, response to the mapping of pyrimidine dimer concentration, is obtained the typical curve of pyrimidine dimer; Testing sample is mixed with the antibody of setting up the typical curve same concentrations, detect mixed liquor and flow through the response that chip produces, itself and typical curve are contrasted, can draw the concentration of pyrimidine dimer in the sample.
2. measure the method for pyrimidine dipolymer content on the DNA chain according to claim 1, it is characterized in that: surpass the testing sample of 500bp for the length of DNA chain, it is interrupted the short chain that forms below the 500pb measure again.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101275885B (en) * | 2007-12-13 | 2010-09-08 | 中国科学技术大学 | Analysis method of solid phase micro-extraction surface plasma resonance on-line joint use |
| CN108530539A (en) * | 2018-03-20 | 2018-09-14 | 北京博雅捷康生物科技有限公司 | The antibody of pyrimidine dimer caused by one group of identification DNA is irradiated by ultraviolet light |
| CN109799337A (en) * | 2019-02-20 | 2019-05-24 | 广东工业大学 | A kind of surface plasmon resonance assay method of quick detection glycocholic acid |
| CN116626189A (en) * | 2023-05-15 | 2023-08-22 | 康龙化成(宁波)科技发展有限公司 | Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5629314A (en) * | 1995-05-15 | 1997-05-13 | Gaskin; Frances C. | Methods and compositions for reducing pyrimidine photoproducts |
| CN1272446C (en) * | 2003-12-05 | 2006-08-30 | 中国科学技术大学 | Process for testing active of DNA optical repairase |
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2006
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101275885B (en) * | 2007-12-13 | 2010-09-08 | 中国科学技术大学 | Analysis method of solid phase micro-extraction surface plasma resonance on-line joint use |
| CN108530539A (en) * | 2018-03-20 | 2018-09-14 | 北京博雅捷康生物科技有限公司 | The antibody of pyrimidine dimer caused by one group of identification DNA is irradiated by ultraviolet light |
| CN108530539B (en) * | 2018-03-20 | 2021-08-13 | 北京博雅捷康生物科技有限公司 | Group of antibodies for recognizing pyrimidine dimers of DNA caused by ultraviolet irradiation |
| CN109799337A (en) * | 2019-02-20 | 2019-05-24 | 广东工业大学 | A kind of surface plasmon resonance assay method of quick detection glycocholic acid |
| CN116626189A (en) * | 2023-05-15 | 2023-08-22 | 康龙化成(宁波)科技发展有限公司 | Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS |
| CN116626189B (en) * | 2023-05-15 | 2024-04-30 | 康龙化成(宁波)科技发展有限公司 | Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS |
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