CN1733935A - Preparation method of DNA microarray chip based on gel fixed nucleic acid - Google Patents
Preparation method of DNA microarray chip based on gel fixed nucleic acid Download PDFInfo
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- CN1733935A CN1733935A CN 200510040597 CN200510040597A CN1733935A CN 1733935 A CN1733935 A CN 1733935A CN 200510040597 CN200510040597 CN 200510040597 CN 200510040597 A CN200510040597 A CN 200510040597A CN 1733935 A CN1733935 A CN 1733935A
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- nucleic acid
- ethylene diamine
- dna microarray
- tetramethyl ethylene
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 57
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 57
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 57
- 238000000018 DNA microarray Methods 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000000758 substrate Substances 0.000 claims abstract description 31
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000007787 solid Substances 0.000 claims abstract description 24
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims abstract description 20
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000004160 Ammonium persulphate Substances 0.000 claims description 14
- 235000019395 ammonium persulphate Nutrition 0.000 claims description 14
- 238000006116 polymerization reaction Methods 0.000 claims description 8
- 239000011521 glass Substances 0.000 claims description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 3
- -1 Tetramethyl Ethylene Diamine Tetramethyl Chemical group 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 238000012986 modification Methods 0.000 claims description 3
- 229910052710 silicon Inorganic materials 0.000 claims description 3
- 239000010703 silicon Substances 0.000 claims description 3
- 230000008020 evaporation Effects 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 239000004033 plastic Substances 0.000 claims description 2
- 229920003023 plastic Polymers 0.000 claims description 2
- WGLLSSPDPJPLOR-UHFFFAOYSA-N tetramethylethylene Natural products CC(C)=C(C)C WGLLSSPDPJPLOR-UHFFFAOYSA-N 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000012935 ammoniumperoxodisulfate Substances 0.000 abstract 2
- 235000011187 glycerol Nutrition 0.000 abstract 2
- 229920001730 Moisture cure polyurethane Polymers 0.000 abstract 1
- 239000000178 monomer Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 34
- 238000009396 hybridization Methods 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 238000002493 microarray Methods 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 239000012496 blank sample Substances 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000001311 chemical methods and process Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 102100021391 Cationic amino acid transporter 3 Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108010001831 LDL receptors Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091006230 SLC7A3 Proteins 0.000 description 1
- 150000003926 acrylamides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 238000011065 in-situ storage Methods 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000003499 nucleic acid array Methods 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
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Abstract
The invention discloses a process for preparing DNA micro array chips based on gel fixed nucleic acid, which comprises mixing acrylamide modified nucleic acid, acrylamide monomer, ammonium peroxodisulfate, glycerin and water into pre-polymer, wherein the concentration of the nucleic acid is between 0.001-100uM, the monomer weight concentration of acrylamide is between 1-30%, the weight concentration of the ammonium peroxodisulfate is between 0.01-5%, the weight concentration of glycerin is between 10-50%, then applying the sample of the prepolymer onto a solid substrate, loading the sample applying substrate into an airtight case containing tetramethylethylenediamine, so as to make the tetramethylethylenediamine volatilize.
Description
Technical field
The present invention relates to a kind of dna microarray chip production method, relate in particular to a kind of dna microarray chip preparation method based on gel fixed nucleic acid.
Background technology
Gene chip is a new and high technology that develops rapidly in life science in recent years.So-called gene chip is exactly silicon chip, slide, the plastic sheet that is fixed with a large amount of gene probes (oligonucleotide probe or cDNA probe) by specific arrangement mode.Biochip technology is to obtain the main means of associated biomolecule information efficiently on a large scale.At present, this technical applications mainly contains gene expression spectrum analysis, new gene discovery, transgenation and polymorphism analysis, genomic library mapping, medical diagnosis on disease and prediction, drug screening, gene sequencing etc.The early 1990s is the main development that begins the various biochips that carry out with the U.S., and less than the time in 10 years, chip technology was developed rapidly, and presented the development peak.The preparation of gene chip at present can be divided into point sample preparation and the synthetic preparation method of original position.With Affymetrix company is that the synthetic preparation method of original position of representative is the most effectual way that makes up the few nucleic acid array of high-density, but make the price of monolithic chip high and can not be widely used owing to it lacks handiness, moreover this method also can't prepare the cDNA chip by pcr amplification.The point sample method has suitable handiness and is fit to great majority research and clinical application.For in for the low-density chip, the point sample method has a production rate faster than original position is synthetic.This method is adopted by most of research unit at present.Determining of the gene chip quality quality of point sample method preparation by following two factors: the one, the carrier of fixed nucleic acid, another is the chemical process that fixed nucleic acid adopted on carrier.Change a kind of method say be exactly the efficient of nucleic acid fixed efficient and nucleic acid hybridization be the accuracy of decision gene chip and two greatest factor of susceptibility.Aspect the carrier use: present widely used carrier is hard carriers such as glass, quartz, and this class carrier is because can only be at its surperficial fixed nucleic acid, and the too high reduction that tends to cause owing to space steric effect hybridization efficiency of nucleic acid constant density.Aspect the chemical fixation method that adopts: no matter hard carrier or gel carriers such as use glass, at present the chemical process of using widely all need the multistep chemical reaction process to carrier activate obtain to be connected with nucleic acid such as aldehyde radical isoreactivity group, process is loaded down with trivial details, length not only consuming time, workload are big, and stability and poor repeatability, make chip quality very unstable.
Summary of the invention
The invention provides a kind of controlled dna microarray chip preparation method based on gel fixed nucleic acid that reacts, the dna microarray chip that is made by the present invention has good homogeneity, repeatability and high accuracy and high susceptibility, and can improve hybridization efficiency.
The present invention adopts following technical scheme:
A kind of dna microarray chip preparation method based on gel fixed nucleic acid, nucleic acid with the acrylamide modification, acrylamide monomer, ammonium persulphate, glycerol and water are mixed into prepolymer, wherein, the concentration of nucleic acid is between 0.001-100uM, the acrylamide monomer weight concentration is between 1-30%, the ammonium persulphate weight concentration is between 0.01-5%, the weight concentration of glycerol is 10-50%, then with the prepolymer point sample on solid substrate, after point sample is intact the point sample substrate placed a closed enclosure that is placed with Tetramethyl Ethylene Diamine, make the Tetramethyl Ethylene Diamine volatilization, the evaporable Tetramethyl Ethylene Diamine contacts with prepolymer lattice point on the solid substrate and makes its polymerization reaction take place, and the nucleic acid on each lattice point is fixed on solid substrate by copolyreaction.
Compared with prior art, the present invention has following advantage:
The present invention utilizes a step not to be subjected to the polyreaction of ectocine to obtain more nucleic acid fixed capacity on three-dimensional porous gel, three-dimensional porous gel can provide the environment that is similar to liquid phase to help hybridization raising hybridization efficiency simultaneously, makes the gene chip of preparation have good accuracy and high susceptibility.The stable C-C key mode of polymeric chemical reaction formation that nucleic acid molecule was not allowed to be subject to ectocine by a step realizes fixing, therefore the fixed nucleic acid that has very high nucleic acid fixed efficiency and acquisition high stability thereof, because the quality of used chemical reagent can obtain by the polymerized in-situ of acrylamide monomer confirming, so good reproducibility.On the other hand because Tetramethyl Ethylene Diamine is the nucleic acid of modifying at acrylamide, acrylamide monomer, glycerol, blended such as ammonium persulphate and water prepolymer point sample makes prepolymer lattice point polymeric by vacuum volatilization behind solid substrate, thereby make this polymerization controlled, prepolymer can polymerization reaction take place before point sample, and the nucleic acid concentration of spotting solution and sample viscosity remain unchanged, and the dna microarray of therefore this nucleic acid fixing means preparation has good homogeneity and repeatability.
Description of drawings
Fig. 1 is a kind of hybridization fluorescent image of oligonucleotide micro-array chip.
Fig. 2 is a kind of hybridization fluorescent image of cDNA micro-array chip.
Embodiment
Embodiment 1
The different nucleic acid that present embodiment is modified acrylamide, respectively with the acrylamide monomer of certain ratio, glycerol, ammonium persulphate and water are mixed into prepolymer, prepolymer with different nucleic acid places on the different positions of point template then, by craft or point sample instrument with nucleic acid prepolymer point sample (utilizing position encoded each the locational nucleic acid molecule of solid substrate that makes is known sample (sequence)) on the different positions of solid substrate, place one to be placed with the airtight moist box of Tetramethyl Ethylene Diamine the point sample substrate after point sample is intact, Tetramethyl Ethylene Diamine volatilization and contact with prepolymer lattice point on the solid substrate and to make its polymerization reaction take place under vacuum action makes the nucleic acid on each lattice point be fixed on solid substrate by copolyreaction.Not homotactic oligonucleotide that (referring to Fig. 1) modifies 5 ' end acrylamide and the blank sample that does not contain nucleic acid are (for example, fully just join with Cy3 pigment flag sequence Cy3-TGC TGT GAG TGA ACC TGC TGT GTT GA-3 ' respectively, middle position mispairing 1, the sequence 5 ' of 2,3 bases-TCA ACA CAG CAG GTT CAC TCA CAG CA-3 ' (P0); 5 '-TCAACA CAG CAG CTT CAC TCA CAG CA-3 ' (P1); 5 '-TCA ACA CAG CAC GAT CAC TCACAG CA-3 ' (P2); 5 '-TCA ACA CAG CAC CAT CAC TCA CAG CA-3 ' (P3) (the letter representation base mismatch of following watchband underscore) respectively with 3% acrylamide monomer, 30% glycerol, 0.1% ammonium persulphate and water are mixed into the prepolymer of 1uM probe, prepolymer with these four kinds of different nucleic acid places on the different positions of point template then, by point sample instrument nucleic acid prepolymer point sample (is represented P respectively on the different positions of solid substrate
0, P
1, P
2, P
3Sequence and each 10 the few nucleic acid lattice point that does not contain the blank sample P of nucleic acid constitute few nucleic acid microarray), place one to be placed with the airtight moist box of Tetramethyl Ethylene Diamine the point sample substrate after point sample is intact, Tetramethyl Ethylene Diamine volatilization and contact with prepolymer lattice point on the solid substrate and to make its polymerization reaction take place under vacuum action makes the nucleic acid on each lattice point be fixed on solid substrate by copolyreaction.Dna microarray chip after fixedly finishing at normal temperatures with Cy3-TGC TGT GAG TGA ACC TGC TGT GTTGA-3 ' sequence hybridization of 10uM 2 hours, obtain the hybridization fluorescent image of micro-array chip with scanner scanning.In Fig. 1, has complete matching sequence (P
0) have a strongest fluorescent signal, the fluorescent signal of mismatch is low than complete matching sequence all, the fluorescence intensity of 1,2,3 bases of its mispairing be respectively fully just joining 51.9%, 2.48% and 0.899%.Clearly strength of signal descends very soon along with the increase of base mispairing number, can distinguish and just join and the single base mismatch sequence.
Embodiment 2
The different PCR products that present embodiment is modified low density lipoprotein receptor gene 14417 site acrylamides, respectively with the acrylamide monomer of certain ratio, glycerol, ammonium persulphate and water are mixed into prepolymer, prepolymer with different nucleic acid places on the different positions of point template then, by craft or point sample instrument with nucleic acid prepolymer point sample on the different positions of solid substrate, be prepared into the cDNA micro-array chip.Its detailed process is: three known 14417 loci polymorphism coronary heart disease patients' fresh blood sample is by anti-phase the primer 5 '-TTT TCA GCA ACT TGG CAT-3 ' amplification of preceding primer 5 '-TAC TAT CCT TCC CAG CTC CT that modifies at 5 ' end acrylamide and unmodified.Contain the 50ng genomic dna in the 50ulPCR system, 1 * PCR damping fluid, 3mM MgCl2,250uM dNTPs, 20pmol primer of 2U Taq archaeal dna polymerase.DNA PCR condition is: 94 ℃ of pre-sex change 5 minutes, 35 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ of circulations of 30 seconds, last 72 ℃ were extended 7 minutes.PCR sample and 3% acrylamide monomer after amplification is finished, 30% glycerol, 1% ammonium persulphate and water are mixed into the prepolymer of about 0.1-0.2uM probe, PCR product and four kinds of prepolymers of blank sample with these three samples place on the different positions of point template then, by point sample instrument with nucleic acid prepolymer point sample on the different positions of solid substrate, place one to be placed with the airtight moist box of Tetramethyl Ethylene Diamine the point sample substrate after point sample is intact, Tetramethyl Ethylene Diamine volatilization and contact with prepolymer lattice point on the solid substrate and to make its polymerization reaction take place under vacuum action makes the nucleic acid on each lattice point be fixed on solid substrate by copolyreaction.CDNA micro-array chip after fixedly finishing at normal temperatures with flag sequence 5 '-Cy3-GGA AAA CAG CCA A-3 ' of 10uM, 5 '-Cy5-GGA AAA GAG CCA A-3 ' sequence hybridization 2 hours, scanning obtains hybridization fluorescent image (Fig. 2).Homozygote 14417C/C provides strong CY3 fluorescent signal (4,5 row among Fig. 2, green fluorescence) among the figure.Homozygote 14417G/G provides strong Cy5 fluorescent signal (7,8 row among Fig. 2, red fluorescence).The fluorescent signal that heterozygous 14417C/G promptly provides Cy3 provides the fluorescent signal of Cy5 again simultaneously, after two kinds of dyestuffs stacks, provides strong yellow fluorescence signal (1,2 row among Fig. 2).
Embodiment 3
A kind of dna microarray chip preparation method based on gel fixed nucleic acid, nucleic acid with the acrylamide modification, acrylamide monomer, ammonium persulphate, glycerol and water are mixed into prepolymer, wherein, the concentration of nucleic acid is between 0.001-100uM, the acrylamide monomer weight concentration is between 1-30%, the ammonium persulphate weight concentration is between 0.01-5%, the weight concentration of glycerol is 10-50%, and the optimum concentration range of nucleic acid is between 0.1-10uM, the optimum concn weight range of acrylamide monomer is between 3-15%, the optimum weight concentration range of ammonium persulphate is between 0.1-1%, specifically, the concentration of nucleic acid can select 0.001,0.1,8,10,50 or 100uM between, the acrylamide monomer weight concentration can select 1%, 3%, 15%, 10%, between 24% or 30%, the ammonium persulphate weight concentration can select 0.01%, 0.1%, 1%, 3%, between 5%, the weight concentration of glycerol can select 10%, 15%, 32%, 42% or 50%, then with the prepolymer point sample on solid substrate, after point sample is intact the point sample substrate placed a closed enclosure that is placed with Tetramethyl Ethylene Diamine, make the Tetramethyl Ethylene Diamine volatilization, the evaporable Tetramethyl Ethylene Diamine contacts and makes its polymerization reaction take place with prepolymer lattice point on the solid substrate, nucleic acid on each lattice point is fixed on solid substrate by copolyreaction, the volatilization mode of described Tetramethyl Ethylene Diamine Tetramethyl Ethylene Diamine can adopt the nature volatilization, evaporation or vacuum volatilization, humidity in the closed enclosure of placement Tetramethyl Ethylene Diamine is 80-100%, solid substrate adopts glass, silicon chip, gel, plastics, a kind of in rubber or the pottery, the nucleic acid that the nucleic acid that the aforesaid propylene acid amides is modified can adopt commercially available acrylamide to modify.
Claims (5)
1, a kind of dna microarray chip preparation method based on gel fixed nucleic acid, it is characterized in that nucleic acid with the acrylamide modification, acrylamide monomer, ammonium persulphate, glycerol and water are mixed into prepolymer, wherein, the concentration of nucleic acid is between 0.001-100uM, the acrylamide monomer weight concentration is between 1-30%, the ammonium persulphate weight concentration is between 0.01-5%, the weight concentration of glycerol is 10-50%, then with the prepolymer point sample on solid substrate, after point sample is intact the point sample substrate placed a closed enclosure that is placed with Tetramethyl Ethylene Diamine, make the Tetramethyl Ethylene Diamine volatilization, the evaporable Tetramethyl Ethylene Diamine contacts with prepolymer lattice point on the solid substrate and makes its polymerization reaction take place, and the nucleic acid on each lattice point is fixed on solid substrate by copolyreaction.
2, the dna microarray chip preparation method based on gel fixed nucleic acid according to claim 1, the optimum concentration range that it is characterized in that nucleic acid is between 0.1-10uM, the optimum concn weight range of acrylamide monomer is between 3-15%, and the optimum weight concentration range of ammonium persulphate is between 0.1-1%.
3, the dna microarray chip preparation method based on gel fixed nucleic acid according to claim 1 is characterized in that the volatilization mode of Tetramethyl Ethylene Diamine Tetramethyl Ethylene Diamine can adopt nature volatilization, evaporation or vacuum volatilization.
4, the dna microarray chip preparation method based on gel fixed nucleic acid according to claim 1, the humidity that it is characterized in that placing in the closed enclosure of Tetramethyl Ethylene Diamine is 80-100%.
5, the dna microarray chip preparation method based on gel fixed nucleic acid according to claim 1 is characterized in that solid substrate adopts a kind of in glass, silicon chip, gel, plastics, rubber or the pottery.
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| CN104927781A (en) * | 2015-04-24 | 2015-09-23 | 王壮昌 | Manufacturing method of gel |
| CN105209638A (en) * | 2013-03-14 | 2015-12-30 | 生命技术公司 | Matrix arrays and methods for making same |
| CN105992815A (en) * | 2013-10-28 | 2016-10-05 | 韩国科学技术研究院 | Porous structure and method for manufacturing same |
| CN107557269A (en) * | 2013-02-26 | 2018-01-09 | 伊鲁米那股份有限公司 | The surface of gel medelling |
| CN113881662A (en) * | 2021-09-09 | 2022-01-04 | 杭州师范大学 | Method for preparing single-stranded DNA (deoxyribonucleic acid) in solid-phase medium through 5' -terminal acrylamide-amidated PCR (polymerase chain reaction) product |
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| US20040253634A1 (en) * | 2001-04-10 | 2004-12-16 | Denong Wang | Novel microarrays and methods of use thereof |
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| CN1271216C (en) * | 2004-05-26 | 2006-08-23 | 中国人民解放军基因工程研究所 | Gene chip surface processing method |
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| CN105209638A (en) * | 2013-03-14 | 2015-12-30 | 生命技术公司 | Matrix arrays and methods for making same |
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| CN105992815A (en) * | 2013-10-28 | 2016-10-05 | 韩国科学技术研究院 | Porous structure and method for manufacturing same |
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