CN1876840A - Multi-copy monomolecular nucleic acid array chip - Google Patents
Multi-copy monomolecular nucleic acid array chip Download PDFInfo
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- CN1876840A CN1876840A CNA2006100399110A CN200610039911A CN1876840A CN 1876840 A CN1876840 A CN 1876840A CN A2006100399110 A CNA2006100399110 A CN A2006100399110A CN 200610039911 A CN200610039911 A CN 200610039911A CN 1876840 A CN1876840 A CN 1876840A
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Abstract
The invention relates to a new monomolecular nucleotide microarray chip with high density and distinguishability. A plurality of polymer high molecule containing nucleotide fragment of this chip can randomly fix on a solid base-plate and can form a monomolecular nucleotide microarray with high density, and can make every array point contain one polymer high molecule, in which the said polymer high molecule contains some similar copies of nucleotide fragment. This chip can be assembled to many kinds of chips used for functional genom study, testing order of complete gene of special types, gene transferring and expression efficiency testing, and testing of kinds of genom DNA modification chart(such as DNA methylation chart). This chip will have great value in the fields of molecular mechanism study in human being life process, genom DNA information decoding, individualized medicine, disease predicting and precaution, medicine developing and individuation.
Description
Technical field
The present invention relates to a kind of high-density nucleic acid microarray chip, this chip can be used for functional genome research, as carry out the detection of gene order-checking, genetic transcription and express spectra of specific species and the detection of range gene group dna modification spectrum (as the dna methylation spectrum), belong to the technical field that biomedical monomolecular nucleic acid array chip is made.
Background technology
Human genome (order-checking) plan is finished, and post genome project has stepped into enforcement.After particularly the Human Genome Project was finished, the related nucleic acid sequence data was exponential growth, and increasing animals and plants, microbial genome sequence are measured, for the research of comparative genomics provides abundant information resources.How a large amount of biomolecules information is analyzed and compared, seek the common molecular mechanism, find new Biological Principles, make biology by experimentally being raised to theory, on the other hand, along with people more in depth are familiar with biological phenomena from molecular level, the clinical medicine practice will produce revolutionary variation.On molecular level, carry out prediction, diagnosis and the treatment of disease, will be familiar with the root that disease produces at all, and will be hopeful basic understanding and treat the major disease that comprises cancer.The change of these biology, medical science, a basic prerequisite is will the genomic information of a large amount of individualities be detected, and to the rapid determination and the evaluation of a large amount of unknown gene sequences.Efficient mensuration of carrying out a large amount of genomes survey row apace will speed up human genome for genomic information and uses, thereby promotes further developing of biology, medical science.
Though obtained great advance the mankind aspect the extensive dna sequencing.Yet, the order-checking of extensive individual whole genome DNA, promptly the genomic dna (including 3,000,000,000 bases) to a large amount of human individuals checks order again, and its cost is still too high.The progress that this has seriously limited functional genome has influence on the application of Human Genome Project achievement in research.We know that the purpose of functional genome research is to be familiar with the biology implication of each nucleic acid in the genome, find diseases predisposing gene.The research approach of functional genome is a comparative genomics, promptly by comparing and analyze genome sequence difference between the different phenotype individualities, the biological significance of each nucleic acid in the decoding genome, discovery feature gene, the molecular genetic information of identification and disease-related.Further, according to genes of individuals group information, realize prediction, prevention and the individualized treatment of disease.Along with the development of functional genome research and people to improving the unremitting pursue of medical treatment ﹠ health level, " individuation medical treatment " begun to enter people's life.For real " individuation medical treatment " this target that realizes, at first to everyone genome be checked order again, find out the difference of Different Individual on dna sequence dna and the dependency of various disease, growth etc.And for present gene order-checking cost, " individuation medical treatment " remains a human remote dream.Therefore,, how fast economically a large amount of individual whole genome to be checked order will be a great challenge, also contain huge business opportunity simultaneously.People are badly in need of developing some quick, cheap individuation gene information detection techniques and are shortened this distance.
Traditional gene order surveying method is being carried out in the improved process, is that the biochip technology of representative arises at the historic moment with the gene chip.The nucleic acid microarray chip is the solid substrate that is assembled with multiple nucleic acid molecule on a kind of surface.The common nucleic acid chip is made up of short ssDNA probe.It by with cell or tissue in the making nucleic acid molecular hybridization that extracts, realize the high throughput testing of gene information.This DNA chip can carry out fast the information nucleic acid in the micro-biological specimen, high-throughput and detect cheaply and analyze, and particularly the characteristics of its high-flux parallel collection bioinformation are that other analytical technology can't be compared at present.The nucleic acid microarray chip has obtained actual application at the analysis of cell and tissue gene express spectra, the examination of Different Individual genome difference, the detection of transgenation and the aspects such as discriminating of multiple pathogenic agent, at aspects such as biological study, biomedical detection, medicament high flux screening and gene sequencings extremely important meaning is arranged.Yet, present nucleic acid microarray biochip technology generally is the oligonucleotide molecules probe array of preparation known array on solid phase carrier, testing gene and oligonucleotide molecules probe array are hybridized, by computer results of hybridization is analyzed, obtained the information of testing gene sequence.Wherein include thousands of identical nucleic acid probes on each array point, to improve the intensity of chip hybridization signal.This has limited the further raising of chip probe density on the one hand; On the other hand, the cost of chip and the complexity of preparation process have greatly been increased; The purity of while sample probe and accuracy and the reliability that fixed efficiency also can influence chip detection.These factors have hindered the result of use of micro-array chip technology greatly, particularly for the use of high-throughout DNA chip.
Therefore, improve and develop new nucleic acid microarray chip, set up novel high-flux sequence method based on biochip to a large amount of genetic information carry out efficiently, the detection of fast and low-cost, to analyze be crucial.
Summary of the invention
Technical problem: the existing problems that the present invention is directed to present biological nucleic acid chip, a kind of monomolecular nucleic acid micro-array chip of multiple copied has been proposed, this chip has the density height, preparation cost is low, method is simple, can be used for the hybridization detection and detect, have crucial application prospect and use value with directly checking order.
Technical scheme: the present invention relates to a kind of novel unit molecule micro-array chip.That is to say, on each array point of chip, only include a polymkeric substance polymer.This polymkeric substance polymer contains the nucleic acid fragment of a plurality of copies, and the sequence of this multiple copied nucleic acid fragment is identical.Be fixed at random at different locational polymkeric substance polymers on this novel unit molecule micro-array chip, the nucleotide sequence on the single polymer molecule on the different positions is inequality.This chip is fixed on one group of polymkeric substance polymer that contains nucleic acid fragment on the solid substrate at random, form a kind of highdensity monomolecular nucleic acid microarray, make to include a polymkeric substance polymer on each array point, and make and contain a plurality of identical nucleic acid fragments copy on the polymkeric substance polymer.This chip will have great application prospect for the aspects such as exploitation of the prediction of the decoding of the Study on Molecular Mechanism of people's vital process, DNA information, individuality medicine, disease and prevention, medicine.
Consisting of of this micro-array chip: on a solid substrate, be fixed with one group of polymkeric substance polymer array, fix one on each point in this polymkeric substance polymer array and contain the unitary polymkeric substance polymer of nucleic acid molecule, the nucleotide sequence on the difference is incomplete same; The nucleic acid molecule unit that the last fixed polymkeric substance polymer of above-mentioned each point is contained is a plurality of copies; Above-mentioned nucleic acid molecule unit is made up of specific nucleic acid fragment and general nucleic acid fragment.
The single-chain nucleic acid of the periodic sequence that the high molecular structure of polymkeric substance is made up of the nucleic acid molecule unit.
By the micro-array chip that the nucleic acid fragment of unit molecule multiple copied is formed, the high molecular structure of its polymkeric substance is the single-chain nucleic acid of the periodic sequence of one section repetitive nucleic acid slice unit composition, as DNA and RNA and other artificial nucleic acids, as PNA, LNA etc.
The high molecular structure of polymkeric substance is the some identical nucleic acid molecule unit and the conjunct polymer molecules of high polymer main chain composition, and wherein, the nucleic acid molecule unit is attached on the high polymer main chain with the form of bifurcated.
Specific nucleic acid fragment on the micro-array chip on each polymkeric substance polymer is a fragment gene group dna sequence dna in the living things system, and the length of this sequence is 5 to 10000 bases; The micro-array chip of being made up of the nucleic acid fragment of unit molecule multiple copied makes up one or more biological genomic dna micro-array chips.
Specific nucleic acid fragment on the micro-array chip on each polymkeric substance polymer is a fragment gene group dna sequence dna in the living things system, the length of this sequence is 5 to 10000 bases, by the micro-array chip that the nucleic acid fragment of unit molecule multiple copied is formed, make up one or more biological open gene dna microarray chips.
The micro-array chip of forming by the nucleic acid fragment of unit molecule multiple copied, the high molecular structure of its polymkeric substance is the some identical nucleic acid fragments and the conjunct polymer molecules of another high polymer main chain (as peptide chain, polyvinyl alcohol etc.) composition, wherein, nucleic acid fragment is attached on the high polymer main chain with the form of bifurcated, be single-chain nucleic acid, as DNA and RNA and other artificial nucleic acids, as PNA, LNA etc.
The micro-array chip of forming by the nucleic acid fragment of unit molecule multiple copied, the high molecular structure of its polymkeric substance is the conjunct polymer molecules that some nucleic acid fragments and another net high-polymer (as agarose, polyacrylamide etc.) are formed, wherein, nucleic acid fragment is attached on the high polymer main chain, be single-chain nucleic acid, as DNA and RNA and other artificial nucleic acids, as PNA, LNA etc.
By the micro-array chip that the nucleic acid fragment of unit molecule multiple copied is formed, the high molecular structure of its polymkeric substance is that some nucleic acid fragments and other microballoon (as agarose, polyacrylamide etc.) are formed.Wherein, nucleic acid fragment is attached on the microsphere surface, is single-chain nucleic acid, as DNA and RNA and other artificial nucleic acids, as PNA, LNA etc.
The micro-array chip of being made up of the nucleic acid fragment of unit molecule multiple copied makes up one or more biological genomic dna micro-array chips, and the specific nucleic acid fragment on the micro-array chip on each polymkeric substance polymer is a fragment gene group dna sequence dna in the living things system.The length of this sequence is 5 to 10000 bases.
By the micro-array chip that the nucleic acid fragment of unit molecule multiple copied is formed, make up that the specific nucleic acid fragment on each polymkeric substance polymer is a fragment gene group dna sequence dna in the living things system on one or more biological open gene dna microarray chip micro-array chips.The length of this sequence is 5 to 10000 bases.
Micro-array chip by the nucleic acid fragment of unit molecule multiple copied is formed makes up the genomic dna micro-array chip.
Beneficial effect:
(1) has only one polymkeric substance polymer on this micro-array chip on each array point.Therefore, the size of each array point can be reduced to the scope of tens nanometers in theory, has so just improved the density of nucleic acid fragment array on the chip greatly.And each array point generally includes thousands of extremely millions of nucleic acid molecule on the existing micro-array chip, and the size of each array point is many at present between several microns and hundreds of microns.For example, the most highdensity nucleic acid microarray chip in the world that the world-renowned manufacturers U.S. Ai Fei company of biochip prepares with the microelectronics photoetching process, the diameter of each array point is 5 microns.With the micro-array chip that common point sample legal system is equipped with, the diameter of its each array point is generally greater than being 50 microns.With the micro-array chip of mini sprinkler preparation, the diameter of its each array point is generally greater than being 100 microns.Therefore, nucleic acid microarray chip of the present invention can have the reticular density of huge nucleic acid microarray on principle.The nucleic acid probe density of unit surface can improve 1,000,000 times.
(2) though going up, each array point of this micro-array chip has only one polymkeric substance polymer, but, the specific nucleic acid fragment that includes a large amount of copies on each polymkeric substance polymer, the nucleic acid fragment of a plurality of copies on the unit molecule can obtain high detection sensitivity equally.Experimental study shows: on single polymkeric substance polymer, can prepare up to ten thousand the segmental copies of specific nucleic acid, can obtain with common micro-array chip on the comparable nucleic acid hybridization signal of array point.Add the raising of present optical detective technology level, present optical detective technology can detect one fluorescence molecule, and therefore, this monomolecular nucleic acid micro-array chip can be applied to the detection of actual sample.
(3) because each array point of this micro-array chip has only a molecule, therefore, the nucleic acid fragment on this microarray point does not exist purifying or pollution problems.In common micro-array chip since the fragment that on an array point, will fix a large amount of same nucleotide sequences as detection probes, the purity of these nucleic acid fragments, and the detection quality of this array point that will directly influence of the pollution in preparation process.Highdensity nucleic acid microarray chip in U.S. Ai Fei company with the preparation of microelectronics photoetching process, because nucleic acid in-situ chemical synthetic productive rate is lower, to have the nucleic acid probe of a large amount of resultant faults on each array point of chip, this will influence the detection accuracy of this chip hybridization to a large extent.
(4) owing to arranging of array point on the common micro-array biochip is pre-designed.Therefore, the position on nucleic acid sequence fragments and the chip is one to one.But, the preparation section complexity of these chips, consuming time and preparation cost height.And in the present invention on the unit molecule micro-array chip nucleic acid polymers polymer on the array point evenly fixedly get at random at one time, the nucleic acid fragment that is assembled on the polymkeric substance polymer is one section dna sequence dna on the known organism genome, carry out the identification of sequence by follow-up combined hybrid detection method or synthetic sequence measurement, by comparing the one-to-one relationship of setting up lattice position on nucleic acid sequence fragments and the chip with original series.Therefore, the unit molecule micro-array chip preparation time that the present invention proposes is short, and method is simple, and cost is low.
The nucleic acid microarray chip of preparation unit molecule multiple copied need with substrate can be silicon, glass, pottery, metal, inorganic or organic solid material such as polymkeric substance, and the molecular film of on these material surfaces, modifying or assembling, its surface can be fine and close, also can be porous.
Description of drawings
The invention will be further described below with reference to accompanying drawing.
The structural representation of a kind of unit molecule multiple copied micro-array chip of forming by single stranded DNA of Fig. 1.A wherein: substrate; B: polymkeric substance polymer array; C: nucleic acid molecule unit; D: polymkeric substance polymer; E: specific nucleic acid fragment; F: general nucleic acid fragment.
The structural representation of a kind of unit molecule multiple copied micro-array chip of forming by organic polymer polymer and nucleic acid molecule polymerization of Fig. 2.A wherein: substrate; B: polymkeric substance polymer array; C: nucleic acid molecule unit; D: polymkeric substance polymer; E: specific nucleic acid fragment; F: general nucleic acid fragment; G: high polymer main chain.
Fig. 3 is combined as example with Nla III restriction endonuclease and Dpn II restriction endonuclease, its two universal joint sequences Design figure.Wherein i:Nla III restriction endonuclease is identified as a little; Ii:Dpn II restriction endonuclease is identified as a little; Iii: universal joint sequence; Iv: enzyme is cut product; V: phosphate group;
The preparation process synoptic diagram of Fig. 4 immobilization unit molecule multiple copied DNA chip.Wherein 1: genomic dna; 2: endonuclease bamhi; 3: universal joint; 4: can hybridize the complementary universal primers with two universal joints, its 5 ' end connected clique of mark (as amino); 5: the restriction endonuclease digestion fragment of cyclisation; The multiple copied single strand dna of 6:RCA expansion; 7: immobilized unit molecule multiple copied single stranded DNA.I: restriction endonuclease digestion; II-III: the connection of universal joint; IV: the universal primer of mark amino and the hybridization of joint; V: the connection of joint makes enzyme cut the product cyclisation; The VI:RCA expansion; VII: unit molecule multiple copied dna fragmentation fixing.
The parallel pcr amplification of Fig. 5 universal primer.A among the figure: the Auele Specific Primer that has general end; B: the universal primer that is marked with the rivet group; C: can with high molecular polymer bonded rivet group; D: genomic dna.
The preparation process synoptic diagram of Fig. 6 prepared with microemulsion reactor unit molecule multiple copied nucleic acid microarray chip.A wherein: substrate; B: polymkeric substance polymer array; C: nucleic acid molecule unit; D: polymkeric substance polymer; E: specific nucleic acid fragment; F: general nucleic acid fragment; G: high polymer main chain; H: the Auele Specific Primer that has general end; I: unit molecule genomic dna; J:PCR reactive component (water); K: oil phase (mineral oil); L: microemulsion system.
Embodiment
● the selection of restriction enzyme site and preparation annular strand genomic DNA fragment
The restriction enzyme digestion of genomic dna: at first select suitable restriction endonuclease (as table one) that genomic dna is carried out enzyme and cut, genome is cut into the sticky end fragment of 50～200bp size by the method for information biology.According to the feature of the end sequence behind each endonuclease digestion, design 2 universal joints simultaneously.Fig. 3 is combined as example with Nla III restriction endonuclease and Dpn II restriction endonuclease, its two universal joint sequences Design figure.The human genome DNA who extracts from blood is after digestion under the different restriction enzymes, and the product of getting 1 μ l detects enzyme and cuts effect 1.5% agarose gel electrophoresis 30 minutes.Then with purification kit with the digestion product purifying, dissolve in the TE damping fluid ,-20 ℃ of preservations are standby.
The design of table 1 restriction endonuclease recognition site sequence and connector end sequence
P-: mark phosphoric acid group mark; Underscore represents that enzyme cuts the end sequence of digestion.
● the preparation of complete genomic unit molecule multiple copied dna fragmentation micro-array chip
The surface amino groups silylation modification of substrate: (A) select the total reflection slide of sizeable low fluorescence background, at 80 ℃ piranha washing lotions (vitriol oil and 30% H
2O
2Mixing in 7: 3 by volume) supersound washing is 2 hours, thoroughly cleans with washing composition then; With deionized-distilled water thoroughly clean once more, dry for standby; (B) silanization is handled: the slide glass in last step washes clean places the acetone soln that contains 2%APTES (3-Aminopro-pyltriethoxysilane) to soak 5～10 minutes, thoroughly clean each twice with acetone, ethanol, deionized-distilled water, nitrogen dries up, and 180 ℃ were toasted 1～2 hour.(C) amination is handled: the slide that silanization is handled places and contains 5% glutaraldehyde (50% aqueous solution, Amresco) soaked 2 hours in the phosphoric acid buffer (0.1M criticizes H7.4), thoroughly clean each 3 times with acetone, ethanol, deionized-distilled water, nitrogen dries up, and 4 ℃ of preservations are standby.(D) all must on the highly sensitive opticmicroscope, carry out fluorescence background behind slide of every processing and detect, in case the interference that the bad fluorescence background that causes of treatment effect detects follow-up single fluorescence molecule.
The joint of genomic DNA fragment connects: the method for the connection of the restriction enzyme digestion of genomic dna and joint primer as shown in Figure 4: (I) genomic dna of Ti Quing (1) passes through the dna fragmentation (2) that above-mentioned different restriction enzymes are digested to 50～200bp length respectively; (II) at T
4Add respectively in the dna ligase reaction system 50nM renaturation become the joint primer (3) of duplex structure and enzyme to cut product (2), (III) 16 ℃ of ligations are after 10 minutes, by the joint primer that electrophoretic method removal does not in short-term connect, the rubber tapping purifying connects product; (IV) add the universal primer (4) of 100nM 5 ' mark amino group in connecting product, 95 ℃ of sex change 5 minutes are cooled to room temperature, and this universal primer can be complementary fully with the joint (3) at two ends, makes enzyme cut product after the renaturation and form a cyclic DNA; (V) will go up the renaturation product that obtains of step at T
4The ligation of dna ligase forms ring-like dna molecular (5) down; (VI) will go up the connection product in step is primer with universal primer (4), with cyclic DNA (5) is masterplate, in Φ 29DNA polymeric enzyme reaction system, carry out dna circle amplified reaction (Rolling Cycle Amplification, RCA), obtain single stranded DNA (6), this DNA chain is the tumor-necrosis factor glycoproteins of cyclic DNA (5).
The preparation (Fig. 4 VII) of unit molecule multi-copy gene group dna fragmentation micro-array chip: (A) with DNA purification kit purifying RCA reaction product, dissolve in carbonate buffer solution (pH 9.0) then, determine the dna molecular concentration of each dilution gradient with ultraviolet spectrophotometer; (B) on the carbonate buffer solution that will dissolve in the RCA product evenly tiles to the surface of 1cm * 1cm in the middle of the total reflection slide of handling, dry back 37 ℃ of aquations naturally and spend the night; At last slide is thoroughly cleaned 2 times respectively with 2 * SSC/0.1%SDS and deionized-distilled water, just be prepared into unit molecule multi-copy gene group dna fragmentation micro-array chip (7).
Microemulsion means that one or more liquid are dispersed in another not miscible liquid with particulate (drop or liquid crystal) form and constitutes the heterogeneous dispersion system with quite stable, because their outward appearances often are emulsus, so be referred to as milk sap.Its principal feature is exactly a Thermodynamically stable, isotropy, and appearance transparent or translucent, the particle diameter even is to micron order.The microemulsion correlation technique is very general in chemistry, and we use characteristics such as its Thermodynamically stable just with in the droplet of biochemical reactions such as pcr amplification as for microemulsion, from but reaction can efficiently carry out fast.
Specific embodiment is as follows:
● the unit molecule Parallel PC R in the microemulsion
The oil phase (mineral wet goods) and the tensio-active agent (Tween that are fit at first selected in the preparation of water in oil microemulsion, SDS, Triton etc.), be water with Parallel PC R amplification system, oil phase and water be by 20: 1 to 1: 1 mixed, prepares microemulsion by vibration or mode such as ultrasonic.The microemulsion of preparation is examined under a microscope its size and uniformity coefficient etc.Ratio and preparation method by control water and oil phase, preparation temperature, conditions such as surfactant concentrations, thereby the microemulsion system of acquisition size homogeneous are further optimized the suitable unit molecule multiple copied amplification system with the little basis of high-polymer molecular of the microemulsion particle diameter for preparing.
The parallel pcr amplification of universal primer is in order to carry out Parallel PC R in microemulsion system, we design and have synthesized a pair of universal primer sequence (Fig. 5), can combine with each end to specific primer sequence, the annealing temperature basically identical of the binding sequence of the Auele Specific Primer in the system is no more than 10 ℃ up and down.One end of this universal primer is to be fixed on the end group of high molecular polymer by certain chemical group (NH2, third rare acid amides, etc.).Macromolecular compound is the link molecule that is fixed in the surface, base when being used for preparing chip.To be marked with the universal primer that group is decided in riveting in the macromolecular compound connection, in microemulsion system, add the Auele Specific Primer amplification that walks abreast, the thermal circulation parameters of microemulsion amplification system is similar to regular-PCR, but its sex change annealing equal time all can shorten to 5-10 second, the preparation of the unit molecule multiple copied nucleic acid that can realize efficient and sensible with regard to increasing in the microemulsion system like this.
● the preparation of unit molecule multiple copied nucleic acid microarray chip
The surface amino groups silylation modification of substrate: (A) select the total reflection slide of sizeable low fluorescence background,, thoroughly clean with washing composition then 80 ℃ piranha washing lotions (vitriol oil mixed with 30% H2O2 in 7: 3 by volume) supersound washing 2 hours; With deionized-distilled water thoroughly clean once more, dry for standby; (B) silanization is handled: the slide glass in last step washes clean places the acetone soln that contains 2%APTES (3-Aminopro-pyltriethoxysilane) to soak 5～10 minutes, thoroughly clean each twice with acetone, ethanol, deionized-distilled water, nitrogen dries up, and 180 ℃ were toasted 1～2 hour.(C) amination is handled: the slide that silanization is handled places and contains 5% glutaraldehyde (50% aqueous solution, Amresco) soaked 2 hours in the phosphoric acid buffer (0.1M criticizes H7.4), thoroughly clean each 3 times with acetone, ethanol, deionized-distilled water, nitrogen dries up, and 4 ℃ of preservations are standby.(D) all must on the highly sensitive opticmicroscope, carry out fluorescence background behind slide of every processing and detect, in case the interference that the bad fluorescence background that causes of treatment effect detects follow-up single fluorescence molecule.
Point sample prepares unit molecule multiple copied nucleic acid microarray chip and at first the above-mentioned unit molecule multiple copied nucleic acid product of micro emulsion method preparation of utilizing is separated from microemulsion system, under the situation of certain example concentration and tensio-active agent existence, centrifugal 5 minutes of 10000～15000g, water phase separated and oil phase, water is assigned to uniformly then and modifies good surface, base (Fig. 6) through diluting with carbonate buffer solution behind the purifying.Thereby be prepared into unit molecule multiple copied nucleic acid microarray chip.
Claims (5)
1. the monomolecular nucleic acid micro-array chip of a multiple copied, it is characterized in that consisting of of this micro-array chip: on a solid substrate (a), be fixed with one group of polymkeric substance polymer array (b), fix a polymkeric substance polymer (d) that contains nucleic acid molecule unit (c) on each point in this polymkeric substance polymer array (b), the nucleotide sequence on the difference is incomplete same; The nucleic acid molecule unit (c) that the last fixed polymkeric substance polymer (d) of above-mentioned each point is contained is a plurality of copies; Above-mentioned nucleic acid molecule unit (c) is made up of specific nucleic acid fragment (e) and general nucleic acid fragment (f).
2. the monomolecular nucleic acid micro-array chip of multiple copied according to claim 1 is characterized in that: the single-chain nucleic acid of the periodic sequence that the structure of polymkeric substance polymer (d) is made up of nucleic acid molecule unit (c).
3. the monomolecular nucleic acid micro-array chip of multiple copied according to claim 1, it is characterized in that: the structure of polymkeric substance polymer (d) is the some identical nucleic acid molecule unit (c) and the conjunct polymer molecules of high polymer main chain (g) composition, wherein, nucleic acid molecule unit (c) is attached on the high polymer main chain with the form of bifurcated.
4. according to the monomolecular nucleic acid micro-array chip of claim 1 or 2 or 3 described multiple copieies, it is characterized in that: the specific nucleic acid fragment (e) on the micro-array chip on each polymkeric substance polymer (d) is a fragment gene group dna sequence dna in the living things system, and the length of this sequence is 5 to 10000 bases; The micro-array chip of being made up of the nucleic acid fragment of unit molecule multiple copied makes up one or more biological genomic dna micro-array chips.
5. the monomolecular nucleic acid micro-array chip of multiple copied according to claim 1, it is characterized in that: the specific nucleic acid fragment (e) on the micro-array chip on each polymkeric substance polymer (d) is a fragment gene group dna sequence dna in the living things system, the length of this sequence is 5 to 10000 bases, by the micro-array chip that the nucleic acid fragment of unit molecule multiple copied is formed, make up one or more biological open gene dna microarray chips.
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| CNA2006100399110A CN1876840A (en) | 2006-04-26 | 2006-04-26 | Multi-copy monomolecular nucleic acid array chip |
| PCT/CN2007/000803 WO2007124651A1 (en) | 2006-04-26 | 2007-03-13 | An array chip of multiple copies of unimolecular nucleic acids |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009135388A1 (en) * | 2008-05-09 | 2009-11-12 | Zhiping Liu | A molecule detecting system |
| CN101948741A (en) * | 2010-09-21 | 2011-01-19 | 东南大学 | Microfluidic gene chip for nucleic acid sequencing |
| CN101413034B (en) * | 2008-11-21 | 2011-02-09 | 东南大学 | Method for preparing molecular cloning chip for high-throughput cloning of nucleic acid molecule |
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| US6284497B1 (en) * | 1998-04-09 | 2001-09-04 | Trustees Of Boston University | Nucleic acid arrays and methods of synthesis |
| EP1907571B1 (en) * | 2005-06-15 | 2017-04-26 | Complete Genomics Inc. | Nucleic acid analysis by random mixtures of non-overlapping fragments |
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- 2007-03-13 WO PCT/CN2007/000803 patent/WO2007124651A1/en active Application Filing
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009135388A1 (en) * | 2008-05-09 | 2009-11-12 | Zhiping Liu | A molecule detecting system |
| CN101413034B (en) * | 2008-11-21 | 2011-02-09 | 东南大学 | Method for preparing molecular cloning chip for high-throughput cloning of nucleic acid molecule |
| CN101948741A (en) * | 2010-09-21 | 2011-01-19 | 东南大学 | Microfluidic gene chip for nucleic acid sequencing |
| CN101948741B (en) * | 2010-09-21 | 2014-02-05 | 东南大学 | Microfluidic gene chip for nucleic acid sequencing |
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| Publication number | Publication date |
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| WO2007124651A1 (en) | 2007-11-08 |
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