CN100590204C - Method for preparing three-dimensional gel micro array chip without excitant - Google Patents
Method for preparing three-dimensional gel micro array chip without excitant Download PDFInfo
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- CN100590204C CN100590204C CN200810020796A CN200810020796A CN100590204C CN 100590204 C CN100590204 C CN 100590204C CN 200810020796 A CN200810020796 A CN 200810020796A CN 200810020796 A CN200810020796 A CN 200810020796A CN 100590204 C CN100590204 C CN 100590204C
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Abstract
The invention discloses a method of preparing three-dimensional gel microarray chip with out stimulating agents, which is characterized in that firstly a pre-polymer can be achieved by mixing water together with nucleic acid modified by an acrylamide group, acrylamide, gel polymerization catalyst and propanetriol; wherein, in the pre-polymer, the concentration of the nucleic acid is ranged from 0.005 to 100 micrometers, and the mass concentration of the acrylamide is ranged from 1 percent to 30 percent; and the mass concentration of the gel polymerization catalyst is ranged from 0.001 percentto 2 percent; and the mass concentration of propanetriol is ranged from 10 percent to 55 percent. The pre-polymer can be spotted on a solid substrate, and after the spotting, the substrate can be heated directly. After the heating, the three-dimensional gel microarray chip can be achieved. The DNA microarray detection chip prepared by the invention has the advantages of higher detection accuracy and sensitivity, excellent repeatability and homogeneity, and low gel background, so that the DNA microarray detection chip can be widely used in scientific research and clinical detection and diagnosis.
Description
One, technical field
The invention belongs to the technology of preparing of dna microarray chip, particularly a kind of fast preparation method that need not the acrylamide gel micro-array chip of exciting agent.
Two, background technology
Prior art: the making method of DNA chip mainly contains point sample method and in-situ synthesis.Compare with in-situ synthesis, the point sample method has mobility strong, prepares characteristics fast, therefore can be used in most scientific research and the clinical detection.The nucleic acid chip no matter which kind of method is prepared, the high-level efficiency of nucleic acid fixed stability and probe hybridization is two principal elements that influence accuracy of purpose signal detection and susceptibility.At present, the method for number of chemical fixed nucleic acid has been arranged, for example, nucleic acid can be fixed on slide, on the substrates of different such as quartz and plastics by after the different chemical process process chip.Yet these methods are often all very low to the fixed efficiency of nucleic acid, need just to reach the detection requirement of signal with a large amount of oligonucleotide of artificial synthetic or by to the fixing of the PCR product of large-scale purification.Gel chip with three-dimensional structure has very strong crystallized ability to nucleic acid, has been used to the preparation of three dimensional gel chip as polyacrylamide gel and sepharose.These two kinds of methods all are to prepare gel-film at substrate surface in advance, thereafter it is mutually crosslinked with gel-film to have amino or aldehyde group modified nucleic acid by certain chemical reaction, yet these two kinds of methods are in the fixed dna process, still need to excite gel, so nucleic acid fixed process seems stable inadequately and convenient with the method for chemical reaction; And the gel chip of preparing has very strong background signal.Rehman etc. have developed and a kind ofly 5 ' end is had the nucleic acid that the acrylamide group modifies directly participate in the polyreaction of acrylamide gel, and this method has further increased the crystallized ability to nucleic acid, (can reach 200fmol/mm
2), and gel is fixing very firm to nucleic acid.In this preparation method, owing to contain catalyzer and exciting agent (both exist simultaneously can quicken cohesion) in the acrylamide pre-polymerization mixture simultaneously, so be difficult to prepare the gel chip of homogeneous.Rubina did not use exciting agent when preparing prepolymer in 2004, prepared the lattice point of homogeneous earlier, adopted uv irradiating polymeric method to separate the gel chip of having prepared homogeneous again.Yet the method for this employing ultraviolet lighting is very big for the destruction of needs fixed nucleic acid, so this method seldom is employed.XPF had improved the Rehman preparation method in 2005, prepared the prepolymer homogeneous microarray that does not contain exciting agent earlier, then was placed on to carry out vacuum in the vessel that fill exciting agent and filter.Because exciting agent has volatilization, when rising when evaporateing into the prepolymer array surface, by to the exciting but the acrylamide polymerization reaction take place of catalyzer in the prepolymer, thereby nucleic acid is fixed in the gel.The gel chip that this method can prepare can be used for carrying out high-throughput nucleic acid and detect.But the main polymerization process of this polymerization process is slower, and exciting agent-Tetramethyl Ethylene Diamine, causes the high background of gel chip easily, and is a kind of poisonous gas with irritating smell.So, be badly in need of a kind of time saving and energy saving, quick, simple and convenient and three dimensional gel new preparation technology in scientific research at present and the medical diagnosis with low background.
Three, summary of the invention
Technical problem: the present invention is directed to above-mentioned technological deficiency, a kind of fast preparation method of the dna microarray chip based on gel fixed nucleic acid is provided, relate in particular to a kind of fast preparation method of micro-array chip of the three dimensional gel that need not exciting agent (Tetramethyl Ethylene Diamine), the dna microarray detection chip that is made by the present invention has high detection accuracy and susceptibility, fabulous advantages such as repeatability, homogeneity and low gel background, can be widely used among the diagnosis of scientific research and clinical detection.That this method for preparing the gel chip has is simple, easy to operate, the preparation time of chip is short and can prevent that chip from suffering advantages such as external source pollution.
Technical scheme: technical solution of the present invention is: a kind of preparation method of three-dimensional gel micro array chip without excitant, preparation process is: at first nucleic acid, acrylamide, gel polymerisation catalyzer, glycerol and the water that the acrylamide group is modified is mixed into prepolymer, wherein, in the prepolymer: the concentration of nucleic acid is at 0.0005～100uM, the acrylamide mass concentration is 1～30%, gel polymerisation catalyst quality concentration is 0.001～2%, and the mass concentration of glycerol is 10～55%; The prepolymer point sample on solid substrate, is directly heated substrate after point sample is finished, promptly get three-dimensional gel micro array chip after heating is finished.The nucleic acid that acrylamide is modified is 5 ' or 3 ' the end modified oligonucleotide that acrylamide is arranged that has through chemosynthesis, or process has the nucleic acid product of the primer of acrylamide modification through polymerase chain reaction (PCR) amplification or rolling circle amplification or other any type of gene amplification gained.Acrylamide is the mixture of acrylamide monomer and methene acrylamide monomer, and the mixing quality ratio of acrylamide monomer and methene acrylamide monomer is 5: 1～50: 1.Solid substrate is hard substrate or soft substrate.The catalyzer of gel polymerisation is ammonium persulphate or riboflavin.The Heating temperature that substrate is heated is 60～120 ℃, and be 1～3 minute heat-up time.Hard substrate: slide, silicon chip, pottery or metal, or soft substrate: plastics, nylon membrane.
Beneficial effect: the gel chip production method that the invention provides a kind of novelty.The inventive method prepares the gel chip and has the following advantages: the accuracy of a. detection nucleic acid and highly sensitive.Because the pre-polymerization object point on the chip has absorbed a large amount of heat at short notice, moisture in the prepolymer is evaporation rapidly, the impellent that evaporation produces can make gel restrain cohesion rapidly, so the fixed nucleic acid concentration in the gel can increase, the final strength of signal that detects also can correspondingly improve, like this accuracy of detection of nucleic acids and highly sensitive will improving.B. fabulous repeatability and homogeneity (seeing Fig. 1 .).C. lower background.Because the process of gel is to adopt the method for heated polymerizable, and gel sticking fluorescence impurity can be effectively avoided in heating, the gel background signal adds to need not to use exciting agent Tetramethyl Ethylene Diamine (can pollute gel raises its background), so can be reduced greatly.D. it is short that this method for preparing the gel chip has preparation time simple, easy to operate, chip.The polymerization of prepolymer only needs can finish 1～3 minute heat-up time.
Four, description of drawings
Fig. 1 is the gel chip that utilizes 12 * 5 arrays that this method prepares.
Five, embodiment
Embodiment 1
A kind of preparation method of three-dimensional gel micro array chip without excitant, preparation process is: at first nucleic acid, acrylamide, gel polymerisation catalyzer, glycerol and the water that the acrylamide group is modified is mixed into prepolymer, wherein, in the prepolymer: the concentration of nucleic acid is at 0.0005uM, the acrylamide mass concentration is 1%, gel polymerisation catalyst quality concentration is 0.001%, and the mass concentration of glycerol is 10%; The prepolymer point sample on solid substrate, is directly heated substrate after point sample is finished, promptly get three-dimensional gel micro array chip after heating is finished.The nucleic acid that acrylamide is modified is 5 ' the end modified oligonucleotide that acrylamide is arranged that has through chemosynthesis, and acrylamide is acrylamide monomer and methene acrylamide monomer, and mass ratio is 50: 1 a mixture.Solid substrate is hard substrate or soft substrate.The catalyzer of gel polymerisation is ammonium persulphate or riboflavin.The Heating temperature that substrate is heated is 60 ℃, and be 1 minute heat-up time.The hard substrate can be slide, silicon chip, ceramic plate or tinsel, also can be soft substrate plastic sheet, nylon membrane.
Embodiment 2
A kind of preparation method of three-dimensional gel micro array chip without excitant, preparation process is: at first nucleic acid, acrylamide, gel polymerisation catalyzer, glycerol and the water that the acrylamide group is modified is mixed into prepolymer, wherein, in the prepolymer: the concentration of nucleic acid is at 100uM, the acrylamide mass concentration is 30%, gel polymerisation catalyst quality concentration is 2%, and the mass concentration of glycerol is 55%; The prepolymer point sample on solid substrate, is directly heated substrate after point sample is finished, promptly get three-dimensional gel micro array chip after heating is finished.The nucleic acid that acrylamide is modified is 3 ' the end modified oligonucleotide that acrylamide is arranged that has through chemosynthesis, and acrylamide is acrylamide monomer and methene acrylamide monomer, and mass ratio is 29: 1 a mixture.Solid substrate is hard substrate or soft substrate.The catalyzer of gel polymerisation is ammonium persulphate or riboflavin.The Heating temperature that substrate is heated is 120 ℃, and be 3 minutes heat-up time.The hard substrate can be slide, silicon chip, ceramic plate or tinsel, also can be soft substrate plastic sheet, nylon membrane.
Embodiment 3
A kind of preparation method of three-dimensional gel micro array chip without excitant, preparation process is: at first nucleic acid, acrylamide, gel polymerisation catalyzer, glycerol and the water that the acrylamide group is modified is mixed into prepolymer, wherein, in the prepolymer: the concentration of nucleic acid is at 50uM, the acrylamide mass concentration is 20%, gel polymerisation catalyst quality concentration is 1%, and the mass concentration of glycerol is 25%; The prepolymer point sample on solid substrate, is directly heated substrate after point sample is finished, promptly get three-dimensional gel micro array chip after heating is finished.The nucleic acid that acrylamide is modified is that process has the nucleic acid product of the primer of acrylamide modification through polymerase chain reaction (PCR) amplification or rolling circle amplification or other any type of gene amplification gained.Acrylamide is acrylamide monomer and methene acrylamide monomer, and mass ratio is 5: 1 a mixture.Solid substrate is hard substrate or soft substrate.The catalyzer of gel polymerisation is ammonium persulphate or riboflavin.The Heating temperature that substrate is heated is 90 ℃, and be 2 minutes heat-up time.The hard substrate can be slide, silicon chip, ceramic plate or tinsel, also can be soft substrate plastic sheet, nylon membrane.
The gel chip that embodiment 4. utilizes this method to prepare carries out somatotype to 500 people's same SNP site simultaneously.
5’-GCTTTCTTGTTTGTTTTCCCTCCTTTACCAY(C/T)
CCAGAAATCCATTTGAGTCTGCTCCTTGT-3’
Y in the sequence (C/T), be one with the transgenation of high blood dense lipoprotein (Homo sapiensdystrobrevin binding protein 1, DTNBP1) a relevant gene locus.Amplify this site one fragment gene sequence that contains of 500 people respectively with PCR by a pair of primer (reverse primer has acrylamide and modifies), directly product and acrylamide, ammonium persulphate, glycerol and the water that 5-10 μ l PCR product or purifying are crossed is mixed into prepolymer, and it is transferred in 384 orifice plates, by the chip point sample instrument 500 sampling points are put into microarray on same slide.Then this chip is placed 80 ℃ of heating to take out after 2 minutes, will be fixed on double-stranded DNA sex change on the gel with NaOH, thereby prepare gel micro-array chip with single stranded DNA.At last with this microarray and a pair of fluorescent detection probe (5 '-Cy5-TTTAC CACCCAGAA-3 ' and 5 '-Cy3-TTTACCATCCAGAA-3 ') hybridization, cannot or hybridizing of mispairing is few because probe of just joining and sequence can be hybridized, just can accurately judge at last by double-colored scanning, and according to the ratio size of each sample Two Colour Fluorescence intensity 500 people's SNP somatotype situation to Cy5 and Cy3 fluorescence.
The gel chip that embodiment 5. utilizes this method to prepare is used for the high-throughput gene sequencing
A general PCR reverse primer is modified with acrylamide, and the primer of another forward is not modified.With above-mentioned reverse primer be mixed into prepolymer with acrylamide, ammonium persulphate, glycerol and water, put on chip.With with example 1. in same heating means prepare the gel micro-array chip that contains amplimer fast.A bigger genomic dna is interrupted with ultrasonic wave, connect same general connexon (with the reverse primer complementation that is fixed in the gel) then at each segmental two ends respectively.Cover on the gel microarray with pcr amplification solution (having contained the aq enzyme, damping fluid, forward primer and dNTP), make in each gel microarray lattice point dna fragmentation of evenly distribute or do not have, then the outer redundant solution of gel lattice point is removed.With paraffin oil the microarray lattice point is sealed again, carry out in 35 circulations of sheet pcr amplification.Then the double-stranded DNA that increases is carried out denaturing treatment with NaOH.The lattice point of each gel microarray just only includes monoclonal dna fragmentation like this.At last the gel dna microarray is used for the SOLEX gene sequencing.
Embodiment 6. utilizes gel chip that this method prepares to the detection that methylates of the CpG island in 800 people P16 intron I districts
Directly 800 people's blood sample is suitably diluted the back as amplification template, carry out pcr amplification with a forward primer and a reverse primer with the modification of acrylamide group.After the amplification with the PCR product with four times dehydrated alcohol precipitation 1 hour, remove supernatant after centrifugal, precipitation is made into 5% acrylamide prepolymer, wherein do not contain TEMED, these sample prepolymers are moved into 384 orifice plates, with chip point sample instrument point sample.Chip is directly carried out 80 ℃ of heating 2 minutes, then with damping fluid flushing one time.Carry out denaturing treatment with NaOH again, and with washed with de-ionized water one time.Use fixed single stranded DNA on the S-WAT process chip again, and wash one time with damping fluid.At last with carrying out image scanning after the detection probe, just can accurately judge the situation that methylates on the CpG island of 800 samples by scan image.
Claims (4)
1. preparation method who need not the three-dimensional gel micro array chip of exciting agent, it is characterized in that preparation process is: at first nucleic acid, acrylamide, gel polymerisation catalyzer, glycerol and the water that the acrylamide group is modified is mixed into prepolymer, wherein, in the prepolymer: the concentration of nucleic acid is at 0.0005～100 μ M, the acrylamide mass concentration is 1～30%, gel polymerisation catalyst quality concentration is 0.001～2%, and the mass concentration of glycerol is 10～55%; The prepolymer point sample on solid substrate, is directly heated substrate after point sample is finished, promptly get three-dimensional gel micro array chip after heating is finished; The nucleic acid that described acrylamide group is modified is 5 ' or 3 ' the end modified oligonucleotide that acrylamide is arranged that has through chemosynthesis, or process has the nucleic acid product of the primer of acrylamide modification through polymerase chain reaction (PCR) amplification or rolling circle amplification or other any type of gene amplification gained; Described acrylamide is the mixture of acrylamide monomer and methene acrylamide monomer, and the mixing quality ratio of acrylamide monomer and methene acrylamide monomer is 5: 1～50: 1; The described Heating temperature that substrate is heated is 60～120 ℃, and be 1～3 minute heat-up time.
2. the preparation method who need not the three-dimensional gel micro array chip of exciting agent according to claim 1 is characterized in that described solid substrate is hard substrate or soft substrate.
3. the preparation method who need not the three-dimensional gel micro array chip of exciting agent according to claim 1 is characterized in that described gel polymerisation catalyzer is ammonium persulphate or riboflavin.
4. the preparation method who need not the three-dimensional gel micro array chip of exciting agent according to claim 2 is characterized in that described hard substrate: slide, silicon chip, pottery or metal, or soft substrate: plastics, nylon membrane.
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| CN102791370A (en) * | 2010-03-17 | 2012-11-21 | 罗伯特·博世有限公司 | Microarray comprising immobilisation particles |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US9512422B2 (en) * | 2013-02-26 | 2016-12-06 | Illumina, Inc. | Gel patterned surfaces |
| US10066260B2 (en) * | 2013-03-14 | 2018-09-04 | Life Technologies Corporation | Matrix arrays and methods for making same |
| CN104263830A (en) * | 2014-09-25 | 2015-01-07 | 徐州医学院 | Method for detecting nucleic acid molecules based on acrylamide gel chip |
| CN114088953B (en) * | 2022-01-19 | 2022-04-15 | 中国中医科学院医学实验中心 | Drug target screening protein chip kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102791370A (en) * | 2010-03-17 | 2012-11-21 | 罗伯特·博世有限公司 | Microarray comprising immobilisation particles |
| CN102791370B (en) * | 2010-03-17 | 2016-08-17 | 罗伯特·博世有限公司 | There is the microarray of fixing particle |
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