CN102936755B - Preparation method of monomolecular nucleic acid chip - Google Patents
Preparation method of monomolecular nucleic acid chip Download PDFInfo
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- CN102936755B CN102936755B CN201210402265.5A CN201210402265A CN102936755B CN 102936755 B CN102936755 B CN 102936755B CN 201210402265 A CN201210402265 A CN 201210402265A CN 102936755 B CN102936755 B CN 102936755B
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Abstract
The invention belongs to the technical field of biology, and discloses a preparation method of a monomolecular nucleic acid chip. According to the invention, a mixture of large amounts of micro-nano beads each carrying a short-chain nucleic acid molecule of a free active group and hollow micro-nano beads is obtained; the mixture is settled to a substrate surface; when the free active groups marked on the nucleic acid molecules are subjected to a connection reaction with the active groups carried on the substrate surface, with a steric hindrance effect of the micro-nano beads, the short-chain nucleic acid molecules are prevented from being too close when fixed towards the substrate, and it is ensured that the spaces between adjacent nucleic acid molecules are no smaller than an optical resolution limit; with external forces, the composition that is not connected with the substrate and the hollow micro-nano beads are separated from the substrate surface; settlement and separation are carried out repeatedly, until a full layer of connected composite is paved on the substrate; and the micro-nano beads are dissociated, such that the monomolecular nucleic acid chip is obtained. The method provided by the invention is fast and simple. With the method, a high-flux monomolecular biochip, in which 95% of sample points can be ensured to be a molecule, can be prepared.
Description
Technical field
The present invention relates to a kind of biochip manufacturing technology, specifically, what relate to is a kind of making method of monomolecular nucleic acid chip.
Background technology
Monomolecular nucleic acid chip, namely each sampling point only has a nucleic acid molecule, the data volume that not only can realize unit surface maximizes, and each molecule is modified rear as " bait " by molecular biology method, acquisition target molecule, both hybridization check single nucleotide polymorphism (SNP), copy number variation (CVN) and the detection such as comparative genome hybridization (aCGH) based on array had been can be used for, also can be used for the order-checking of DNA synthesis method, connection method order-checking etc., s-generation sequencing technologies is converted to third generation sequencing technologies, other multiple single molecule studies can also be used for.But the manufacture difficulty of monomolecular nucleic acid chip is large, traditional preparation method is that solution is laid, and has very large randomness, namely has many nucleic acid molecule overlapping and cannot observe, some substrate not having sample, cause limited area of detection to waste.So, up to the present, also more not gratifying technology.
Through finding existing literature search, S.Quake seminar in 2007 has delivered at APPLIED PHYSICS LETTERS and has been entitled as " High density single molecule surface patterning with colloidal epitaxy " DNA molecular is evenly tied to diameter at nano level colloidal solid, DNA nano-array chip is prepared through the effect of colloidal solid epitaxy, but because the DNA molecular number be tied on each particle is a lot, the chip obtained only is had an appointment 1/3 sampling point be unit molecule, other sampling points are polymolecular.Therefore, the method can not ensure it each sampling point is " unit molecule " DNA chip only have a DNA and unit molecule.Therefore, the technical problem to be solved in the present invention is to provide fast, simple preparation guarantees that the sampling point of 95% is the high-throughput unit molecule biochip of a molecule.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of making method of monomolecular nucleic acid chip, by spacing and the control of purity of unit molecule sampling point, set up the manufacturing technology system of high-density, multifunctional single molecular nucleic acid chip, for SNP, CVN and aCGH detection and unique DNA order-checking etc.
For achieving the above object, present invention employs following technical scheme:
Prepare a making method for monomolecular nucleic acid chip, the method comprises the steps:
The first step, get and respectively carry the micro-nano bead (hereinafter referred to as object mixture) of the short-chain nucleic acids molecule of a free active group and empty micro-nano bead mixture in a large number, this mixture is deposited to substrate surface, and this substrate surface carries active group;
Second step, the active group generation ligation that the active group that nucleic acid molecule marks and substrate surface carry, utilize the sterically hindered effect of micro-nano bead limit short-chain nucleic acids molecule basad fixing time too close, ensure that adjacent nucleic acid molecule spacing is not less than optical resolution limit;
3rd step, the object mixture utilizing external force to make not to be connected with substrate and empty micro-nano bead depart from substrate surface, sedimentation and disengaging so repeatedly, until substrate is paved with the object mixture that one deck connects;
4th step, the micro-nano bead be connected with substrate of dissociating, obtains monomolecular nucleic acid chip.
Preferably, the micro-nano bead (object mixture) of the described short-chain nucleic acids molecule with a free active group, prepare in accordance with the following methods:
Step one, the nucleic acid two places being marked with different activities group is dissolved in connection damping fluid, obtains solution I;
Step 2, is connected damping fluid by surface-coated can being suspended in the micro-nano bead that ligation occurs one of two kinds of active groups on nucleic acid, obtains solution II;
Step 3, solution I and solution II to be mixed in setting ratio in nucleic acid molecule number and micro-nano bead number and stirs, corresponding active group on nucleic acid and the active group in micro-nano bead are fully reacted, nucleic acid is tied in micro-nano bead, obtain object mixture.
Described micro-nano bead is monomolecular nucleic acid launch vehicle, and its material can be silicon, magneticsubstance or polymer etc., its diameter in nanometer to micron dimension.
Described nucleic acid molecule can be thymus nucleic acid (DNA), Yeast Nucleic Acid (RNA) and peptide nucleic acid(PNA) (PNA).
Described short-chain nucleic acids is compared with micro-nano bead diameter, the little magnitude or less of its contour length.
Described substrate can be sheet glass, silicon chip and sheet mica etc.
Described sedimentation reaches complex deposits to substrate surface by gravity, magnetic force, centrifugal force and electrical forces etc.
Described steric effect, when object mixture is routed to substrate surface, because length nucleic acid is very short, the nucleic acid only having micro-nano bead to contact on substratel sub-fraction sphere can touch substrate surface and connect, and can guarantee that adjacent nucleic acid molecule spacing is not less than optical resolution limit.
The active group of described nucleic acid marking and micro-nano bead and the coating active group of substrate, be the active group pair that immune response can occur, directly or indirectly form covalent linkage, reach the object that nucleic acid is connected with micro-nano bead and substrate, every active group that can realize this object to, as: biotin-avidin, digoxin-anti digoxin antibody, amino-succinimide, amino-epoxy resin, amino-carboxyl, amino-aldehyde radical, sulfydryl-epoxy resin, sulfydryl-maleimide, sulfydryl-sulfydryl, ketone group-azanol, ketone group-hydrazides, hydrazides-aldehyde radical, hydrazides-carboxyl etc., the active group mark that its amplifying nucleic acid can not react mutually with two, micro-nano bead is with being coated to the active group of an active group generation ligation on nucleic acid, substrate is with marking with the active group of another active group generation ligation on nucleic acid.
Described nucleic acid two place mark active group, can at interval certain number base and nucleic acid two ends.When nucleic acid is DNA, according to the position that its sequences Design and active group mark, being fixed to the DNA molecular of substrate, can be the loop-stem structure DNA of linear ssdna, end flush end or sticky end, the double-stranded DNA of also can be one or both ends be flush end or sticky end; These special sequences and structure, may be used for acquisition target molecular dna or RNA.
Described external force, can be shake, rotate, also can be fluid flushing.
The described micro-nano bead be connected with substrate of dissociating, being adopt zymetology, physics, also can be the method for chemistry, makes micro-nano bead depart from substrate surface, realizes the fixing of object nucleic acid molecule.As utilized the corresponding DNA restriction endonuclease cutting DNA of the selectivity restriction enzyme site of DNA, reach and connect substrate DNA fragmentation and the disengaging being connected micro-nano bead DNA fragmentation, also double-stranded DNA can be unwind by the method for heating, the micro-nano bead and the substrate that make to be connected to DNA two strands depart from, also double-stranded DNA can be unwind with causing the chemical reagent of nucleic acid molecule sex change, as alkaline solution, methyl alcohol, ethanol, urea and methane amide etc., realize being separated of micro-nano bead and substrate.Suprabasil micro-nano bead is cleaned, through reclaiming and after purifying, can reusing with solution.
The nucleic acid molecule number that described micro-nano bead is carried refers to the maximum value calculating acquisition according to the expection unit molecule purity on nano-beads diameter, Poisson formula and chip.If nano-beads diameter is 700nm, for ensureing that chip sampling point spacing is for being not less than 500nm, length of nucleic acid molecule is the longest is 42nt, when the expection unit molecule purity on chip is 98%, and nucleic acid molecule Tiao Shuo≤6 that nano-beads is carried.If micron pearl diameter is 2000nm, length of nucleic acid molecule is 42nt, when the expection unit molecule purity on chip is 80%, and nucleic acid molecule Tiao Shuo≤28 that nano-beads is carried.
Described nucleic acid molecule number mixes in setting ratio with micro-nano bead number, be according to Poisson regularity of distribution P=λ ^k/ (k! E^ λ), wherein k is nucleic acid molecule number, and λ is the ratio of nucleic acid molecule number and micro-nano bead number before mixing, and P is a probability micro-nano bead connecting simultaneously k bar DNA.
Compared with prior art, the present invention has following beneficial effect:
Compared with domestic and international similar chip, the single-molecule manipulation techniques that the present invention adopts, technique is simple, the object mixture built is reusable, be suitable for the preparation of large-scale unit molecule chip, the ratio of unit molecule sampling point can reach more than 95%, and external technology only has 37%, sampling point density can reach 600-700nm, close to optical limit, as adopted 4 look fluorescence, a slide glass prepared by sedimentation repeatedly/electrotransfer method can carry up to 3,500,000,000 sampling points, can be used for SNP, CNV and aCGH detection and monomolecular nucleic acid order-checking etc.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is monomolecular nucleic acid chip manufacturing schematic diagram of the present invention;
In figure: 1. micro-nano bead; 2. active group; 3. monomolecular nucleic acid; 4. fixing adjacent nucleic acid closest range; 5. below horizontal line for nucleic acid molecule can touch the sphere of substrate.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, the condition that conveniently conditioned disjunction is advised according to manufacturers is usually carried out.
As shown in Figure 1, object mixture in following examples, nucleic acid molecule number mixes by a certain percentage with micro-nano bead number, be according to Poisson regularity of distribution P=λ ^k/ (k! E^ λ), wherein k is nucleic acid molecule number, and λ is the ratio of nucleic acid molecule number and micro-nano bead number before mixing, and P is a probability micro-nano bead connecting simultaneously k bar DNA.In order to the sampling point ensureing in DNA chip is unit molecule at high proportion, the probability simultaneously connecting >=2 DNA in theory of probability can be used to be set in sphere (Fig. 1) that nucleic acid molecule can touch substrate is not more than preset value X, the ratio of unit molecule sampling point can be realized for (1-X) × 100/%, simultaneously the diameter of micro-nano bead and length of nucleic acid molecule in Binding experiment, obtain the number k that a ball can connect at most nucleic acid molecule.The probability setting connection >=k bar nucleic acid molecule simultaneously in a micro-nano bead is not more than 0.01(P value), the front nucleic acid molecule number of mixing can be obtained: the ratio λ value of micro-nano bead number.
Embodiment 1
Step one, oneself's pairing and the single strand dna formed containing the loop-stem structure of DNA endonuclease recognized site can be dissolved in the 42nd bit base sulfydryl modification of length 52 Nucleotide (nt), 5 ' end and connect damping fluid with amido modified, obtain solution I;
Step 2, is suspended in connection damping fluid by the silicon nano-beads of the diameter 700nm of surface-coated carboxyl, obtains solution II;
Step 3, solution I and solution II are mixed into reaction vessel in the ratio of DNA molecular number and silicon nano-beads number 2:1 and stir, carboxyl on amino on DNA and nanometer silica bead is fully reacted, is tied to DNA bar number≤6 on a silica bead, obtain object mixture.
Step 4, is deposited to the coating silicon chip surface of LC-SMCC by object mixture by electrical forces, makes the maleimide generation ligation that the sulfydryl that DNA molecular marks is coated to silicon chip surface.
Step 5, by rotational response container, makes the mixture that is not connected with silicon chip and empty nanometer silica bead depart from surface of glass slide, repeating step four, sedimentation and disengaging so repeatedly, until silicon chip is paved with the mixture that one deck connects.
Step 6, utilize restriction endonuclease to cut the selectivity restriction enzyme site of loop-stem structure DNA, buffer solution for cleaning removes nanometer silica bead, obtains monomolecular nucleic acid chip.
Implementation result: the unit molecule chip that the present embodiment is laid, unit molecule sampling point ratio can be made to reach 98%, and nearest sampling point spacing is for being not less than 500nm.
Embodiment 2
Step one, is dissolved in 5 ' of a chain in the double-stranded DNA of length 42 base pair (bp) end with the short chain double chain DNA molecule of 5 ' the end biotin modification of sulfydryl modification, another chain and connects damping fluid, obtain solution I;
Step 2, is suspended in connection damping fluid by the diameter 700nm nanometer silica bead of surface amination, obtains solution II;
Step 3, solution I and solution II to be mixed in reaction vessel than the ratio for 7:1 in DNA molecular number and silica bead number and to stir, amino on sulfydryl on DNA and nanometer silica bead is fully reacted, makes DNA bar number≤14 on a silica bead, obtain object mixture.
Step 4, is deposited to the surface of glass slide of coating avidin by object mixture by gravity, make the avidin generation ligation that vitamin H that DNA molecular marks and surface of glass slide are carried.
Step 5, by rotational response container, makes the mixture that is not connected with slide and empty nanometer silica bead depart from surface of glass slide, repeating step four, sedimentation and disengaging so repeatedly, until slide is paved with the mixture that one deck connects.
Step 6, with the urea-denatured double-stranded DNA of 6-8M, buffer solution for cleaning removes nanometer silica bead, obtains monomolecular nucleic acid chip.
Implementation result: the unique DNA chip that the present embodiment is laid, unit molecule sampling point ratio can reach 80%, and nearest sampling point spacing is not less than 500nm.
Embodiment 3
Step one, the heteroduplex DNA by length 172bp: 5 ' the end digoxin modification of the DNA chain in RNA, 5 ' end biotin modification of RNA chain, be dissolved in connection damping fluid, obtain solution I;
Step 2, is suspended in connection damping fluid by the diameter 1000nm nanometer magnetic bead of surface-coated anti digoxin antibody, obtains solution II;
Step 3, solution I and solution II to be mixed in reaction vessel than the ratio for 1:2 in heteroduplex number and magnetic bead number and to stir, digoxin on DNA and the anti digoxin antibody on magnetic bead are fully reacted, is He acid Fen≤3 on each magnetic bead, obtains object mixture.
Step 4, by the surface of glass slide of object mixture magnetic settlement to coating avidin, makes the anti-chain biotin protein generation ligation that the vitamin H that RNA molecule marks is coated to surface of glass slide.
Step 5, by shake reaction vessel, makes the mixture that is not connected with slide and empty nanometer magnetic bead depart from surface of glass slide, repeating step four, sedimentation and disengaging so repeatedly, until slide is paved with the mixture of one deck connection.
Step 6, with 0.15M NaOH sex change heteroduplex nucleic acid, makes magnetic bead and slide depart from, and buffer solution for cleaning removes magnetic bead, obtains unit molecule RNA chip.
Implementation result: the unit molecule chip that the present embodiment is laid, unit molecule sampling point ratio can be made to reach 98%, and nearest sampling point spacing is for being not less than 500nm.
Embodiment 4
Step one, is dissolved in 5 ' end with the compound molecule that biotin modification, length 172nt DNA, 3 ' end are hybridized with it by 20 base PNA and connects damping fluid, obtain solution I;
Step 2, is suspended in connection damping fluid by the diameter 1000nm pipe/polyhenylethylene nano pearl (PS pearl) of surface-coated avidin, obtains solution II;
Step 3, solution I and solution II to be mixed in reaction vessel than the ratio for 2:1 in compound molecule number and PS pearl number and to stir, avidin on vitamin H on compound molecule and PS pearl is fully reacted, Tiao Shuo≤6 of compound molecule on each PS pearl, obtain object mixture.
Step 4, is deposited to the mica sheet surface of coating succinimide by object mixture centrifugal force, make the succinimide generation ligation that the primary amino group on pna molecule is coating with mica sheet surface.
Step 5, by wash buffer surface, makes the mixture that is not connected with sheet mica and empty PS pearl depart from mica sheet surface, repeating step four, sedimentation and disengaging so repeatedly, until sheet mica is paved with the mixture of one deck connection.
Step 6, at 94 DEG C, denaturation temperature makes compound molecule unwind, and PS pearl and sheet mica are departed from, and buffer solution for cleaning removes PS pearl, obtains unit molecule PNA chip.
Implementation result: the unique DNA chip that the present embodiment is laid, unit molecule sampling point ratio can reach 80%, and nearest sampling point spacing is not less than 500nm.
Embodiment 5
Step one, is dissolved in 5 ' end of 5 ' of a chain in the double-stranded DNA of length 42bp end sulfydryl modification, another chain with amido modified short chain double chain DNA molecule and connects damping fluid, obtain solution I;
Step 2, is suspended in connection damping fluid by the diameter 2000nm micron silica bead of surperficial sulfhydrylation, obtains solution II;
Step 3, solution I and solution II to be mixed in reaction vessel than the ratio for 18:1 in DNA molecular number and silica bead number and to stir, sulfydryl on sulfydryl on DNA and micron silica bead is fully reacted, makes DNA bar number≤28 on a silica bead, obtain object mixture.
Step 4, is deposited to the surface of glass slide of coating lsothiocyanates by object mixture by gravity, make the lsothiocyanates generation ligation that amino that DNA molecular marks and surface of glass slide are carried.
Step 5, by shake reaction vessel, makes the mixture that is not connected with slide and empty micron silica bead depart from surface of glass slide, repeating step four, sedimentation and disengaging so repeatedly, until slide is paved with the mixture of one deck connection.
Step 6, uses formamide denaturation double-stranded DNA, and buffer solution for cleaning removes micron silica bead, obtains monomolecular nucleic acid chip.
Implementation result: the unique DNA chip that the present embodiment is laid, unit molecule sampling point ratio can reach 80%, and nearest sampling point spacing is not less than 500nm.
It should be noted that, above embodiment is section Example of the present invention, portion of techniques etc. in conversion above-described embodiment, the active group of the micro-nano bead material such as described in summary of the invention, nucleic acid molecule type, substrate, subsidence style, nucleic acid marking and micro-nano bead and the coating active group kind of substrate etc., the present invention can realize, and can reach above-mentioned effect.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. prepare the making method of monomolecular nucleic acid chip for one kind, it is characterized in that, the method is after obtaining the micro-nano bead and object mixture and empty micro-nano bead mixture respectively carrying the short-chain nucleic acids molecule of a free active group in a large number, mixture is deposited to substrate surface, when the active group generation ligation that the active group making nucleic acid molecule marks and substrate surface carry, utilize the sterically hindered effect of micro-nano bead limit short-chain nucleic acids molecule basad fixing time too close, ensure that adjacent nucleic acid molecule spacing is not less than optical resolution limit, the object mixture utilizing external force to make not to be connected with substrate and empty micro-nano bead depart from substrate surface, so sedimentation and disengaging repeatedly, until substrate is paved with the object mixture that one deck connects, finally, to dissociate the micro-nano bead be connected with substrate, obtain monomolecular nucleic acid chip,
The nucleic acid molecule number that described micro-nano bead is carried refers to the maximum value calculating acquisition according to the expection unit molecule purity on nano-beads diameter, Poisson formula and chip; If nano-beads diameter is 700nm, for ensureing that chip sampling point spacing is for being not less than 500nm, length of nucleic acid molecule is the longest is 42nt, when the expection unit molecule purity on chip is 98%, and nucleic acid molecule Tiao Shuo≤6 that nano-beads is carried; If micron pearl diameter is 2000nm, length of nucleic acid molecule is 42nt, when the expection unit molecule purity on chip is 80%, and nucleic acid molecule Tiao Shuo≤28 that nano-beads is carried.
2. the making method of monomolecular nucleic acid chip according to claim 1, is characterized in that, described object mixture, prepares in accordance with the following methods:
The first step, the nucleic acid two places being marked with different activities group is dissolved in connection damping fluid, obtains solution I;
Second step, is connected damping fluid by surface-coated can being suspended in the micro-nano bead that ligation occurs one of two kinds of active groups on nucleic acid, obtains solution II;
3rd step, solution I and solution II to be mixed in setting ratio in nucleic acid molecule number and micro-nano bead number and stirs, corresponding active group on nucleic acid and the active group in micro-nano bead are fully reacted, nucleic acid is tied in micro-nano bead, obtain object mixture.
3. the making method of monomolecular nucleic acid chip according to claim 1, is characterized in that, described nucleic acid molecule number mixes in setting ratio with micro-nano bead number, be according to Poisson regularity of distribution P=λ ^k/ (k! E^ λ), wherein k is nucleic acid molecule number, and λ is the ratio of nucleic acid molecule number and micro-nano bead number before mixing, and P is a probability micro-nano bead connecting simultaneously k bar DNA.
4. the making method of monomolecular nucleic acid chip according to claim 3, it is characterized in that, in order to the sampling point ensureing in DNA chip is unit molecule at high proportion, the probability simultaneously connecting >=2 DNA in use theory of probability to be set in sphere that nucleic acid molecule can touch substrate is not more than preset value X, namely the ratio of unit molecule sampling point is realized for (1-X) 100%, simultaneously in conjunction with diameter and the length of nucleic acid molecule of micro-nano bead, obtain the number k that a micro-nano bead can connect at most nucleic acid molecule, the probability setting connection >=k bar nucleic acid molecule simultaneously in a micro-nano bead is not more than 0.01, namely the front nucleic acid molecule number of mixing is obtained: the ratio λ value of micro-nano bead number.
5. the making method of the monomolecular nucleic acid chip according to any one of claim 1-4, it is characterized in that, described nucleic acid molecule, is thymus nucleic acid DNA, RNA (ribonucleic acid) and peptide nucleic acid(PNA) PNA, compared with micro-nano bead diameter, the little magnitude or less of its contour length; When two place's mark active groups, interval certain number base marks, or is marked at nucleic acid two ends; When nucleic acid is DNA, according to the position that its sequences Design and active group mark, be fixed to the DNA molecular of substrate, or the loop-stem structure DNA of linear ssdna, end flush end or sticky end, or one or both ends are the double-stranded DNA of flush end or sticky end; These special sequences and structure, for acquisition target molecular dna or RNA.
6. the making method of the monomolecular nucleic acid chip according to any one of claim 1-4, it is characterized in that, the active group of described nucleic acid marking and micro-nano bead and the coating active group of substrate, be the active group pair that immune response can occur, directly or indirectly form covalent linkage, reach the object that nucleic acid is connected with micro-nano bead and substrate; Every active group that can realize this object to, the active group mark that its amplifying nucleic acid can not react mutually with two, micro-nano bead is with being coated to the active group of an active group generation ligation on nucleic acid, and substrate is with marking with the active group of another active group generation ligation on nucleic acid.
7. the making method of monomolecular nucleic acid chip according to claim 6, it is characterized in that, described active group is to comprising: biotin-avidin, digoxin-anti digoxin antibody, amino-succinimide, amino-epoxy resin, amino-carboxyl, amino-aldehyde radical, sulfydryl-epoxy resin, sulfydryl-maleimide, sulfydryl-sulfydryl, ketone group-azanol, ketone group-hydrazides, hydrazides-aldehyde radical, hydrazides-carboxyl.
8. the making method of the monomolecular nucleic acid chip according to any one of claim 1-4, it is characterized in that, described micro-nano bead of dissociating adopts the method for zymetology, physics or chemistry, micro-nano bead is made to depart from substrate surface, realize the fixing of object nucleic acid molecule, comprise and utilize the corresponding DNA restriction endonuclease cutting DNA of the selectivity restriction enzyme site of DNA, reach and connect substrate DNA fragmentation and the disengaging being connected micro-nano bead DNA fragmentation; By the method for heating, double-stranded DNA is unwind, the micro-nano bead and the substrate that make to be connected to DNA two strands depart from; With the chemical reagent of nucleic acid molecule sex change can be caused to make double-stranded DNA unwind, realize being separated of micro-nano bead and substrate.
9. the making method of the monomolecular nucleic acid chip according to any one of claim 1-4, is characterized in that, described micro-nano bead, is monomolecular nucleic acid launch vehicle, and its material is silicon, magneticsubstance or polymer, its diameter in nanometer to micron dimension; Described substrate is sheet glass, silicon chip or sheet mica.
10. the making method of the monomolecular nucleic acid chip according to any one of claim 1-4, is characterized in that, described is deposited to surface, base by mixture, is reach complex deposits to substrate surface by gravity, magnetic force, centrifugal force and electrical forces; Described external force is shake, rotates or fluid flushing.
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| CN103088435A (en) * | 2013-01-30 | 2013-05-08 | 中国科学技术大学 | Renewable biotin binding protein chip and preparation method thereof |
| CN103305603B (en) * | 2013-03-26 | 2015-05-20 | 上海交通大学 | Method for preparing single polymer carried by single micro/nano bead by single-molecule bioreactor |
| CN103194371B (en) * | 2013-03-26 | 2014-12-24 | 上海交通大学 | Separation and collection device for biological compound |
| CN103194370B (en) * | 2013-03-26 | 2014-12-24 | 上海交通大学 | Device for preparing and enriching single micro-nano bead carried simple-root polymer molecule |
| CN110283888B (en) * | 2013-08-19 | 2021-06-22 | 卓异生物公司 | Assays for single molecule detection and uses thereof |
| CN104535769B (en) * | 2014-12-12 | 2016-07-06 | 上海交通大学 | Through the method that high density nanometer hole prepares biomacromolecule unimolecule chip |
| CN113528623A (en) * | 2015-02-18 | 2021-10-22 | 卓异生物公司 | Assays for single molecule detection and uses thereof |
| US20190284552A1 (en) * | 2016-05-26 | 2019-09-19 | Singular Bio, Inc. | Arrays for Single Molecule Detection and Uses Thereof |
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| CN101619353A (en) * | 2009-08-13 | 2010-01-06 | 上海交通大学 | Method for linking monomolecular DNA to single magnetic bead |
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