CN1379114A - Process for preparing microarray chip of double-stranded nucleic acid - Google Patents
Process for preparing microarray chip of double-stranded nucleic acid Download PDFInfo
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- CN1379114A CN1379114A CN 02112780 CN02112780A CN1379114A CN 1379114 A CN1379114 A CN 1379114A CN 02112780 CN02112780 CN 02112780 CN 02112780 A CN02112780 A CN 02112780A CN 1379114 A CN1379114 A CN 1379114A
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Abstract
A process for preparing double-stranded nucleic acid microarray chip from single-stranded oligonucleotide includes such steps as transferring the single-stranded oligonucleotide fragment containing two reverse complementary sequence pieces at 3' terminal to the surface of solid substrate, linking its 5' terminal to the surface of solid substrate to generate the single-stranded oligonucleotide microarray, annealing the said two reverse complementary sequence to generate hairpin structure, and relying on polymerizing reaction of nucleic acid polymerase to fill up the remaining single-stranded part so as to make up the said microarray chip. It can be used to detect the mutual action between the double-stranded nucleic acid (dsDNA, for example) and the protein.
Description
One, technical field:
The present invention is the method for preparing micro-array chip in the biomedical sector, especially a kind of method that is prepared microarray chip of double-stranded nucleic acid by single stranded oligonucleotide.
Two, background technology:
Biochip mainly is meant the microarray that biologically active substances such as the nucleic acid assembled, protein, cell, small tissue constitute on solid support.Applying biochip can realize treating the having that it's too late and how much carry out accurately of materials such as target nucleic acid molecules, protein molecule, cell, small tissue in the sample product, fast, large information capacity detects.Detected characteristics with microminiaturization, high-throughput and automatization.Biochip has extremely important using value meaning on biological detection, medical science detection and medical diagnosis on disease, drug screening and gene sequencing, therefore be subjected to extensive concern and investment, make it become global novel industry, i.e. a biochip industry.
At present the research and development kind of biochip is tending towards variation gradually, comprise initial exploitation and widespread use gene chip, powerful pressing protein chip, the cell chip with life characteristic and organization chip just come onto stage.The detection that realizes on two aspects genetic expression transcribed and translated to gene chip and protein chip can, in the research of genome and protein group, become extremely important technology platform, simultaneously biomedical sectors such as medical clinic applications, new medicament screen, efficacy analysis have been produced revolutionary impact.
Go up from constituting, gene chip and protein chip are made of single strand dna oligonucleotide molecule and protein molecule respectively, therefore gene chip can only rely on hybridization between the nucleic acid molecule detect target single strand dna in the sample behind nucleic acid amplification have that it's too late what, protein chip mainly the identification by protein target molecule in protein and the sample and combine the detection protein target molecule how much have that it's too late.Therefore gene chip can not detect protein, protein chip also is difficult to detect DNA, this has limited utilization DNA chip directly to proteinic detection, and DNA and proteinic identification and interact is kept and expression of gene is brought into play important role in regulating in genome stability.It mainly is that gel shift is tested (gel shift mobility assay) that tradition is used for the ordinary method of researching DNA and protein interaction in the molecular biology, but this method is big to laboratory sample quantity demand, test length consuming time, analysis efficiency is low, and has the harm of radio-labeling to experimenter and environment.
Microarray chip of double-stranded nucleic acid is a kind of biochip.Microarray chip of double-stranded nucleic acid can overcome the single-chain nucleic acid microarray can not detect proteinic defective, because conjugated protein usually identification of DNA and bonded are double-stranded DNAs, takes this to play a significant role in the expression of gene regulation and control.The preparation of microarray chip of double-stranded nucleic acid is the key of this chip application.Lockhart etc. just proposed to prepare method (the US patent No.5 of double chain DNA probe molecule as far back as 1996 on solid-phase media, 556,752), its basic skills is that the oligonucleotide of two sections base complementrities utilization link molecule is linked together, one section is fixed on the solid-phase media, make the oligonucleotide renaturation of two sections base complementrities by annealing, form the double-strandednucleic acid unit molecule that has prominent ring, be used to detect the DNA binding biomolecules; This method is owing to two sections oligonucleotide of chemosynthesis, so production efficiency is low, and cost is higher.Bulyk etc. proposed the microarray chip of double-stranded nucleic acid notion the earliest in 1999, and utilization nucleic acid polymerase primer extension method prepares double-strandednucleic acid microarray (US.Pat.6,326,489).It to the effect that at first passes through the synthesizing single-stranded oligonucleotide microarray of photo etched mask original position on slide, every single stranded oligonucleotide of synthetic is near 3 of slide ' contain the universal primer anneal sequence, after single stranded oligonucleotide microarray and the universal primer annealing, carry out the polysaccharase primer extension reaction, the synthetic second chain nucleic acid, thus the double-strandednucleic acid microarray formed.The shortcoming of this method is to synthesize the long oligonucleotide that contains primer binding site on the solid-phase media surface, is unfavorable for reducing production costs.Because being subjected to protection of Intellectual Property Rights can not be in China's application of commercializing.
Three, summary of the invention:
(1) goal of the invention
The purpose of this invention is to provide a kind of method of being convenient to prepare the double-strandednucleic acid microarray, and the method that preparation cost is lower, the more simple complementary single-chain nucleic acid renaturation of the base mispairing rate of chip double-strandednucleic acid prepares double-strandednucleic acid is low in the common equipment condition.Can be used for the process for preparing microarray chip of double-stranded nucleic acid of the bigger single base mutation microarray chip of double-stranded nucleic acid of productive value
(2) technical scheme
The present invention is the preparation method of microarray chip of double-stranded nucleic acid, it is characterized in that this preparation method comprises the steps: that (a) is connected to the solid support surface with the fixed single stranded oligonucleotide fragment of chemosynthesis by its 5 ' end, forms the single stranded oligonucleotide microarray; Fixed single stranded oligonucleotide fragment contains two sections reverse complementary base sequences thereof at its freedom 3 ' end; (b) its freedom 3 of fixed single stranded oligonucleotide fragment ' two sections reverse complementary base sequences thereof annealing of end form a hairpin structure at single stranded oligonucleotide fragment 3 ' end; (c) nucleic acid polymerase is that primer carries out the Nucleotide polyreaction with the hairpin structure, fill and lead up the segmental residue strand of fixed single stranded oligonucleotide part, at the synthetic complete double chain acid molecule of support surface, make the single stranded oligonucleotide microarray become the double-strandednucleic acid microarray; Its freedom 3 of fixed single stranded oligonucleotide fragment ' end is not connected with the solid support surface, is in free state; Fixed single stranded oligonucleotide fragment 3 ' two sections reverse complementary base sequences thereof annealing of end form hairpin structure; This hairpin structure is one section short double chain oligonucleotide fragment, and it only has 3 of an open state ' end, and 3 ' hold to be hydroxyl; The hairpin structure that annealing forms serves as the primer of nucleic acid polymerization reaction, discerned combination by nucleic acid polymerase, start the Nucleotide polyreaction, fill and lead up the segmental residue strand of fixed single stranded oligonucleotide part, thereby change the single stranded oligonucleotide microarray of preparation into the double chain oligonucleotide microarray; Fixed single stranded oligonucleotide fragment comprises Yeast Nucleic Acid and thymus nucleic acid two class nucleic acid; The nucleic acid polymerase that is used to carry out the Nucleotide polyreaction comprises ribonucleic acid polymerase, deoxyribonucleic acid polymerase and ThermoScript II; The Nucleotide that is used to carry out the Nucleotide polyreaction comprises all ribonucleotides, all deoxyribonucleotides and special minor nucleotide; The solid support that is used for fixing single stranded oligonucleotide comprises the material that has rigidity and semi-rigid surface.
(3) technique effect
The process for preparing microarray chip of double-stranded nucleic acid that the present invention proposes has 3 important value in stdn, mass production: the one, and the reverse complementary sequence annealing that the process for preparing microarray chip of double-stranded nucleic acid that proposes relies on fixed single-chain nucleic acid itself to contain forms hairpin structure provides primer, by the synthetic double-strandednucleic acid of polysaccharase primer extension, than second long molecule primer or longer complementary single-chain nucleic acid of complete sequence and fixed microarray single-chain nucleic acid annealing hybridization, reduced production cost without any need for extra composition sequence; During the double-strandednucleic acid microarray that the present invention proposes is produced, only need the chemosynthesis single-chain nucleic acid to get final product, all the other complementary nucleic acids are just synthetic by cheap polysaccharase hairpin structure primer extension reaction, and this double-strandednucleic acid microarray production to longer sequence is even more important; The 2nd, the present invention proposes hairpin structure provides primer to pass through the method that the primer-oligomerization enzyme extends synthetic double-strandednucleic acid, because the fidelity of nucleic acid polymerase, carrying out property be than the height of chemosynthesis, therefore for long or to contain the nucleic acid fragment of degenerate core thuja acid synthetic significant; The 3rd, the double-strandednucleic acid microarray synthetic method that the present invention proposes can guarantee base mispairing rate extremely low in the synthetic double-strandednucleic acid, and this has very important value for the single base mutation microarray chip of double-stranded nucleic acid that production has more large-scale commerce value; This method can overcome the synthetic cover of high price and but can not have the critical defect of the microarray chip of double-stranded nucleic acid of single base mutation feature by the method production of hybridization renaturation at sheet synthetic microarray single-chain nucleic acid complementary single-chain nucleic acid.
Protein-bonded research especially has important value to the process for preparing microarray chip of double-stranded nucleic acid that the present invention proposes to DNA.DNA and protein interactions genome stability keep and gene expression regulation in bringing into play important role, SDBP particularly, though this proteinoid only account for the nucleus total protein less than 0.1%, but this proteinoid usually is to comprise that the regulatory gene of transcription factor is with certain space-time, the important trans-acting factor of quantity expression, they are by own special structure such as leucine zipper, sequence-specific double-stranded DNA identification combination in the genomes such as zinc fingers, accurately regulate a class function Expression of Related Genes, therefore to the research in this proteinoid and specific recognition bonded DNA site thereof at molecular biology, genomics reaches in the proteomics that has become the primary study object at present and all occupies important status.Yet the methods such as the gel shift experiment that the research in this proteinoid and specific recognition bonded DNA site thereof is developed in traditional molecular biology at present, DNA enzyme I foot printing test, do not have a kind of high-throughout stdn screening method, and the single stranded DNA gene chip and the protein chip that have developed at present all are difficult to carry out the efficient screening and the evaluation of this proteinoid.Double-stranded DNA microarray that the present invention proposes and preparation method thereof is exactly to be used for filling up this defective specially, carries out screening in the nucleus whole protein class range research method of SDBP and DNA recognition site thereof.The foundation of this research platform has important value for the following problem of research.
The one, double-strandednucleic acid microarray that utilization the present invention proposes and preparation method thereof can go out the conjugated protein a complete set of single base mutation double-stranded DNA of the low density micro-array chip as NF-kB of certain sequence-specific double-stranded DNA by gene chip sample applying instrument stdn efficient production, this chip reacts as NF-kB with the corresponding sequence-specific double-stranded DNA of fluorophor mark is conjugated protein, by of the analysis of obtaining of stdn gene chip scanning instrument to hybridization signal, the base that just can obtain conjugated protein identification of this DNA and bonded sequence-specific double-stranded DNA site constitutes characteristics, finds that those participate in the base and the action rule thereof of DNA and protein identification and bonded most critical.
The 2nd, double-strandednucleic acid microarray that utilization the present invention proposes and preparation method thereof can by the machine point sample prepare contain 6～10 bases the high-density double-stranded DNA micro-array chip of the function sequence that might make up, as contain 8 bases might the combination function sequence the double-stranded DNA microarray of 66536 double-chain probes, contain 10 bases might the combination function sequence the double-stranded DNA microarray of 1048576 double-chain probes, use the full nucleus albumen of such double-stranded DNA micro-array chip and certain cell to react, just can obtain all possible SDBP is listed as its dna binding sequence on the chip in the full nucleus albumen of certain cell identification and combining information, thereby scan the DNA site of the conjugated protein identification of all possible DNA that exists on the chip, scan simultaneously with chip on the DNA site all possible DNA of bonded conjugated protein.This is conjugated protein and the conjugated protein DNA recognition site of DNA is very valuable for high flux screening DNA.
The 3rd, double-strandednucleic acid microarray that utilization the present invention proposes and preparation method thereof can be prepared suitable density by point sample but contain the double-stranded DNA micro-array chip of the conjugated protein binding site dna fragmentation of having discovered at present of all DNA, by be in the Different Organs tissue, different developmental phases, full nucleus protein hybridization reaction such as the cell of different pathological status etc., just can obtain with the double-stranded DNA micro-array chip on all DNA bonded DNA conjugated protein at the Different Organs tissue, different developmental phases, expressing information in the cells such as different pathological status, thus their function and mutual relationship disclosed; This data message is as if the gene expression information in conjunction with conventional expression type gene chip, just can obtain all possible gene of the conjugated protein participation regulation and control of certain DNA, and different genes can be carried out function and sort out, make the mutual relationship of genetic expression in order, this has very important value for great biological questions such as research biological growth, pathology and molecular medicine diagnosis, treatment, new drug developments.
Double-strandednucleic acid microarray (chip) preparation method of the uniqueness that the present invention foundes can synthesize the double-strandednucleic acid microarray accurately, efficiently, economically on solid support such as slide, this double-strandednucleic acid microarray not only its preparation basis is compatible fully with domestic and international gene chip technology of preparing, and compatible fully with the reaction of general gene expression chip, test set in application, improved the simplicity of operation of this invention product.
Therefore the present invention proposes a kind of new process for preparing microarray chip of double-stranded nucleic acid, this method need be in sheet original position synthetic complex process, only need single stranded nucleic acid molecule of chemosynthesis, be made into the single-chain nucleic acid microarray by point sample, again by a renaturation and polymerase elongation reaction, just the single-chain nucleic acid microarray can be transformed into the double-strandednucleic acid microarray, not only be convenient to preparation double-strandednucleic acid microarray under the common facility condition, and preparation cost is lower, the method that the more complementary single-chain nucleic acid renaturation of the base mispairing rate of chip double-strandednucleic acid prepares double-strandednucleic acid is low.The most important thing is that this double-strandednucleic acid microarray preparation method can be used for the bigger single base mutation microarray chip of double-stranded nucleic acid of productive value, and that this is the method that relies on merely complementary single-chain nucleic acid renaturation to prepare double-strandednucleic acid is indeterminable.
Four, description of drawings:
Fig. 1 is the single stranded oligonucleotide fragment that will be fixed on the solid support surface after the chemosynthesis.Comprising reverse complementary sequence S1, S2.
Fig. 2 be by 5 ' be fixed on the single stranded oligonucleotide fragment on solid support surface.
Fig. 3 is 3 ' oppositely complementary base sequences thereof annealing forms hairpin structures and nucleic acid polymerase in conjunction with 3 ' hydroxyl.
Fig. 4 is that the nucleic acid polymerase extension is filled and led up the segmental residue strand of single stranded oligonucleotide part.
Fig. 5 is that the nucleic acid polymerase extension stops the double-strandednucleic acid array that the back forms.
Fig. 6 is the basic building block that is used for the single stranded oligonucleotide of synthetic dsdna microarray.
Fig. 7 is that the synthetic enzyme that reaches of Cy3 mark dATP infiltration double-stranded DNA microarray is tested conscientiously.
Fig. 8 is that Cy3 mark NF-kB and double-stranded DNA microarray hybridization and enzyme are tested conscientiously.
Five, embodiment
1, the preparation of solid support
Solid support with the double-strandednucleic acid microarray of the present invention that fixes comprises biological and abiotic, organic and inorganic material, perhaps the mixture of these materials.These materials may exist with the state of particulate, wire, throw out, gel, thin slice, pipeline, spheroid, container, kapillary, liner, section, film, disc, slide, but shape easily such as employing disc preferably, slide, globule, bead, disk.The solid support material preferably has at least one side fully smooth.Solid support also can be selected other surface shapes, recessed or protruding zone is arranged so that carry out building-up reactions in the above as the surface.Sometimes select suitable solid support in order to suitable optical absorption characteristics to be provided, the gelinite, polymkeric substance that as upholder may be the silicon of glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modification of polymeric LB film, functionalization or any kind of are as (gathering) tetrafluoroethylene ((poly) tetrafluoroethylene), (gathering) vinylidene fluoride ((poly) vinylidendifluoride), polystyrene (polystyrene), polycarbonate (polycarbonate) or their combination; Similar groups such as active group such as carboxyl, amino, hydroxyl, thiol group are preferably contained on the solid support surface.Preferably optical clear and the Si--OH surface such of solid support surface in addition just like silicon materials surfaces.
2, the chemosynthesis of single stranded oligonucleotide
Use needed nucleotide sequence design single stranded oligonucleotide according to concrete, and comprise the reverse complementary base sequences thereof (Fig. 1: S1, S2) of two sections suitable length, adopt the synthetic single stranded oligonucleotide that designs of commercialization solid state chemistry synthetic program at its 3 ' end.Chemical reaction can be carried out with the solid support surface active groups and the chemical group that connects together in single stranded oligonucleotide 5 ' end connection in synthetic synthetic back, as amino etc., 3 ' hold to be oh group.As Fig. 1.
3, chain oligonucleotide fixing on the solid support surface
The solid support surface is transferred to by suitable mode such as machine, craft or electrophoresis in the synthetic back of solid state chemistry synthetic single stranded oligonucleotide, and single stranded oligonucleotide is connected to the solid support surface under proper condition.As Fig. 2.
4, single stranded oligonucleotide 3 ' end reverse complementary sequence annealing renaturation forms hairpin structure
In 3 fixedly the solid support of single stranded oligonucleotide in redistilled water 100 ℃ boiled 5 minutes, after nitrogen dries up, add the nucleic acid hybridization damping fluid of optimal temperature preheating, at optimal temperature hybridization appropriate time.As Fig. 3.
5, oligonucleotide hairpin structure 3 ' terminal hydroxy group combines with nucleic acid polymerase
After the solid support of handling in 4 is removed hybridization buffer with the simple washing of redistilled water, add required Nucleotide and nucleic acid polymerase after, at optimal temperature reaction appropriate time.As Fig. 4.
6, stop polyreaction, form the complete double chain DNA fragment that comprises former hairpin structure
Solid support in 5 after the polysaccharase polyreaction is used 2 * SSD, 0.1%SDS and 0.2 * SSD, 0.1%SDS washing successively, simply washs with redistilled water at last, and it is standby to put 4 ℃ of preservations.As Fig. 5.
Design and the synthetic single stranded oligonucleotide dna fragmentation that contains following member composition carry out the feasibility confirmatory experiment of microarray chip of double-stranded nucleic acid of the present invention:
5 ' NH
2----the other sequence of 4 base near-ends----4 base HaeIII restriction enzyme sites----the conjugated protein NF-kB binding site of 10 base DNA (function sequence)----other sequence----5 base hairpin structure sequences 1 (reverse complementary sequence 1)----2 irrelevant base intervening sequences (hair fastener ring sequence)----5 base hairpin structure sequence 2 (reverse complementary sequence 2)----3 ' hydroxyls (OH) of 4 base far-ends.
Use this single stranded oligonucleotide to carry out following two experiments.
Fluorescent mark of the present invention (Cy3) ATP infiltration method levies in kind polysaccharase is synthetic to second chain
(1) with top synthetic single stranded oligonucleotide by 5 ' NH
2Point sample is connected on the slide that the silanization glutaraldehyde handles and forms 3 * 3 arrays, and sealing is soaked with sodium borohydride in 4 ℃ of backs of spending the night, and washs slide with redistilled water again.
(2) will boil in the slide boiling water 5 minutes, dry up after being cooled to 55 ℃, rapidly at the nucleic acid hybridization damping fluid of 5 ℃ of preheatings of single stranded oligonucleotide microarray place Dropwise 5, after cover glass covers, the inherent 55 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(3) remove hybridization buffer with the simple washing of redistilled water, slide oligonucleotide microarray place drips the polyreaction damping fluid that contains dGTP, dCTP, dTTP, Cy3-dATP and archaeal dna polymerase (Klenow enzyme), after cover glass covers, the built-in 37 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(4) remove the polyreaction damping fluid with the simple washing of redistilled water, dry up behind the slide and scan in the gene chip scanning instrument.
(5) after the slide after will scanning takes out, drip HaeIII and enzyme cutting buffering liquid thereof at slide oligonucleotide microarray place, after cover glass covers, the inherent 37 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(6) remove the polyreaction damping fluid with the simple washing of redistilled water, dry up behind the slide and scan in the gene chip scanning instrument.
Fluorescent mark (Cy3) NF-kB combines double-stranded DNA microarray and the conjugated protein identification bonded of the DNA feasibility that confirms the present invention's preparation with the double-stranded DNA microarray
(1) with top synthetic single stranded oligonucleotide by 5 ' NH
2Point sample is connected on the slide that the silanization glutaraldehyde handles and forms 3 * 3 arrays, and sealing is soaked with sodium borohydride in 4 ℃ of backs of spending the night, and washs slide with redistilled water again.
(2) will boil in the slide boiling water 5 minutes, dry up after being cooled to 55 ℃, rapidly at the nucleic acid hybridization damping fluid of 5 ℃ of preheatings of single stranded oligonucleotide microarray place Dropwise 5, after cover glass covers, the inherent 55 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(3) remove hybridization buffer with the simple washing of redistilled water, drip the polyreaction damping fluid that contains dGTP, dCTP, dTTP, dATP and archaeal dna polymerase (Klenow enzyme) at slide oligonucleotide microarray place, after cover glass covers, the built-in 37 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(4) remove the polyreaction damping fluid with the simple washing of redistilled water, drip NF-kB and the DNA binding buffer liquid that contains the Cy3 mark at slide oligonucleotide microarray place behind the drying slide, after cover glass covers, the built-in 25 ℃ of water bath heat preservations of slide moisture releasing box 30 minutes.
(5) remove the association reaction damping fluid with the simple washing of redistilled water, dry up behind the slide and scan in the gene chip scanning instrument.
(6) after the slide after will scanning takes out, drip HaeIII and enzyme cutting buffering liquid thereof at slide oligonucleotide microarray place, after cover glass covers, the inherent 37 ℃ of water bath heat preservations of slide moisture releasing box 2 hours.
(7) remove the polyreaction damping fluid with the simple washing of redistilled water, dry up behind the slide and scan in the gene chip scanning instrument.
Claims (8)
1, a kind of process for preparing microarray chip of double-stranded nucleic acid is characterized in that this preparation method comprises the steps:
(a) the single stranded oligonucleotide fragment with chemosynthesis is connected to the solid support surface by its 5 ' end, forms the single stranded oligonucleotide microarray; Fixed single stranded oligonucleotide fragment contains two sections reverse complementary base sequences thereof at its freedom 3 ' end;
(b) its freedom 3 of fixed single stranded oligonucleotide fragment ' two sections reverse complementary base sequences thereof annealing of end form a hairpin structure at single stranded oligonucleotide fragment 3 ' end;
(c) nucleic acid polymerase is that primer carries out the Nucleotide polyreaction with the hairpin structure, fill and lead up the segmental residue strand of fixed single stranded oligonucleotide part, at the synthetic complete double chain acid molecule of support surface, make the single stranded oligonucleotide microarray become the double-strandednucleic acid microarray.
2, process for preparing microarray chip of double-stranded nucleic acid according to claim 1 is characterized in that its freedom 3 of fixed single stranded oligonucleotide fragment ' end is not connected with the solid support surface, is in free state.
3, process for preparing microarray chip of double-stranded nucleic acid according to claim 1 is characterized in that fixed single stranded oligonucleotide fragment 3 ' end contains two sections reverse complementary base sequences thereof, their formation hairpin structures of can annealing; This hairpin structure is one section short double chain oligonucleotide fragment, and it only has 3 of an open state ' end, and 3 ' hold to be hydroxyl.
4, according to claim 1 or 3 described process for preparing microarray chip of double-stranded nucleic acid, the hairpin structure that forms that it is characterized in that annealing can serve as the primer of nucleic acid polymerization reaction, can be discerned combination by nucleic acid polymerase, start the Nucleotide polyreaction, fill and lead up the segmental residue strand of fixed single stranded oligonucleotide part, thereby change the single stranded oligonucleotide microarray of preparation into the double chain oligonucleotide microarray.
5, process for preparing microarray chip of double-stranded nucleic acid according to claim 1 is characterized in that fixed single stranded oligonucleotide fragment comprises Yeast Nucleic Acid and thymus nucleic acid two class nucleic acid.
6, process for preparing microarray chip of double-stranded nucleic acid according to claim 1, the nucleic acid polymerase that it is characterized in that being used to carrying out the Nucleotide polyreaction comprises ribonucleic acid polymerase, deoxyribonucleic acid polymerase and ThermoScript II.
7, process for preparing microarray chip of double-stranded nucleic acid according to claim 1, the Nucleotide that it is characterized in that being used to carrying out the Nucleotide polyreaction comprises all ribonucleotides, all deoxyribonucleotides and special minor nucleotide.
8, process for preparing microarray chip of double-stranded nucleic acid according to claim 1 is characterized in that the solid support that is used for fixing single stranded oligonucleotide comprises the material that has rigidity and semi-rigid surface.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100396791C (en) * | 2004-09-17 | 2008-06-25 | 北京大学 | Method for detecting oligomeric nucleotide through intercalating dye fluorescent resonant energy transfer molecule |
| CN100396790C (en) * | 2004-09-17 | 2008-06-25 | 北京大学 | Solution identification and surface addressing protein chip and its preparing and detecting method |
| CN101236209B (en) * | 2007-09-03 | 2013-03-20 | 博奥生物有限公司 | Method for detecting if interaction between nucleic acid conjugated protein -target protein exist based on biological chip |
| CN106460032A (en) * | 2013-12-05 | 2017-02-22 | 生捷科技控股公司 | Fabrication of patterned arrays |
-
2002
- 2002-03-20 CN CNB021127808A patent/CN1142292C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100396791C (en) * | 2004-09-17 | 2008-06-25 | 北京大学 | Method for detecting oligomeric nucleotide through intercalating dye fluorescent resonant energy transfer molecule |
| CN100396790C (en) * | 2004-09-17 | 2008-06-25 | 北京大学 | Solution identification and surface addressing protein chip and its preparing and detecting method |
| CN101236209B (en) * | 2007-09-03 | 2013-03-20 | 博奥生物有限公司 | Method for detecting if interaction between nucleic acid conjugated protein -target protein exist based on biological chip |
| CN106460032A (en) * | 2013-12-05 | 2017-02-22 | 生捷科技控股公司 | Fabrication of patterned arrays |
| CN106460032B (en) * | 2013-12-05 | 2019-12-24 | 生捷科技控股公司 | Preparation of patterned arrays |
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