CN1400315A - Gene chip high-flux quick detection technique - Google Patents
Gene chip high-flux quick detection technique Download PDFInfo
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- CN1400315A CN1400315A CN 01123528 CN01123528A CN1400315A CN 1400315 A CN1400315 A CN 1400315A CN 01123528 CN01123528 CN 01123528 CN 01123528 A CN01123528 A CN 01123528A CN 1400315 A CN1400315 A CN 1400315A
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Abstract
The present invention discloses a universal detection technique for gene chip and its application. Said ivnention greatly simplifies the operative steps for detecting gene chip, can effectively prevent mutual interference of tens of hundreds of primer pairs when high flux detection is made so as to implement high flux rapid detection of gene chip, and the stability, specificity, sensitiivty and reproducibility of detection result are good.
Description
The present invention relates to new gene chip high-flux quick detection technique and their application.
One. the current situation of gene chip:
The U.S. continues and carries out after the Human Genome Project, the formal promotor gene chip plan in 1998, and the state-run Ministry of Health of the U.S., Ministry of Energy, the Ministry of Commerce, the Ministry of Justice, Department of Defense, Central Office of Information etc. have all participated in this project.Stanford University, Massachusetts Institute of Technology and the state-run laboratory of part such as Argonne Oakridge have also participated in the research and development of this project simultaneously.Univ cambridge uk, Eurasian company are being engaged in the research in this field.It is interested that world big drug firm especially is used for gene pleiomorphism, disease-related, genomic medicine exploitation and fields such as synthetic or natural drug screening to biochip technology, all set up or set up oneself chipset and technology.
At home, though starting slightly a little later, the R﹠D work of chip is all being done by scientific research institutions such as Tsing-Hua University, Fudan University, Beijing Academy of Military Sciences, Nanjing Southeast China University.The work that these units carried out mainly is with the research of gene chip as gene function, that is to say chip is used as " chip gene expression profile ", the work that it is done is to filter out the gene with certain function from hundreds of bar, several ten thousand, tens0000 genes.But the imperfection of gene chip screening effect has determined it can only use for scientific research at present, still can not enter clinical use.The domestic company that also has is developing the gene chip that clinical diagnosis is used at present, but owing to all adopt following experiment path, loaded down with trivial details and sensitivity is not high, repeatability is bad, low-density gene chip still can be used, highdensity gene chip then is reacted to hybridization from PCR and all is difficult to control, and the gene chip that causes clinical diagnosis to be used is difficult to enter clinical practice.
At present all DNA chip technologies all comprise four bare bones: the amplification of the structure of DNA array (little ordering), sample (DNA or RNA) and the detection of mark, hybridization and results of hybridization and read.
The structure of DNA array (little ordering): prepare chip at present and mainly adopt the method for surface chemistry or the method for combinatorial chemistry to handle solid phase carrier (sheet glass or silicon chip), dna fragmentation is arranged on the solid phase carrier in order.More because of the solid phase carrier kind, the preparation method also is not quite similar, but can be divided into two big classes basically: a class is original position synthetic (in situ Synthesis); One class is synthetic back crosslinked (post-synthesis attachment).The synthetic oligonucleotide that is applicable to of original position; Synthetic back is crosslinked uses large fragment DNA more, also is used for oligonucleotide sometimes, even mRNA.
The amplification of sample DNA or RNA and mark: the biomolecules mixture that biological sample is very complicated often, except that the minority particular sample, generally can not directly be detected by chip, sample must be handled.The DNA/mRNA sample that obtains from blood, living tissue or other sample must amplification be read sensitivity to improve, and detect beacon with fluorescein, isotropic substance, vitamin H on the tense marker etc., but this process operation gets up to have certain degree of difficulty.Such as the interference that thousands of normal genes are arranged in a cancer cells, the detection of heterozygosis oncogene and to it efficiently, amplification specifically is not a nothing the matter just.Because when polymerase chain reaction (PCR) increases in general solution, there is the situation of other different dna fragmentation and target fragment competition primer.Another kind of situation is that gene chip will detect a lot of target dnas usually, and when pcr amplification, if exist simultaneously in the reaction system much to primer, then interfere with each other and compete between primer and the template DNA, reaction product often is not a target dna, if further carry out chip detection, then false positive results may appear.
Hybridization: hybridization be in the DNA chip technology except that little ordering of DNA and sample amplification a most important step, the degree that it is complicated and the control of actual conditions are decided by the length of the dna fragmentation on the chip and the purposes of chip itself.If detection of expression need high salt concentration, low temperature and long lasting (often requiring to spend the night) during hybridization, but the preciseness requirement is then lower.If detect whether sudden change is arranged, because of relating to the mispairing of single base, so needs at short notice under (several hours), less salt, the hot conditions high preciseness hybridize.
The detection of results of hybridization and reading: at present target dna is adopted fluorescent marker method mostly, and read the result with scanning confocal microscope or laser confocal scanning according to fluorescent signal power of each hybridization point.Its advantage is a good reproducibility, and shortcoming is that sensitivity is relatively low.For this reason, people are studying multiple alternative method, as: mass spectroscopy, chemoluminescence and photoconductive fiber, the detection of diode square formation, latex agglutination test, directly change in electrical charge detection or the like.Wherein the most promising working as, push away mass spectroscopy, but owing to also there are some problems in the chemosynthesis of probe, mass spectroscopy is used generally not as good as fluorescent marker method.
In the experiment of reality, be four above-mentioned steps because gene chip all adopts, caused gene chip to delay to enter large-scale practical application.
In order to solve the many mutual interference mutually to primer and template in the PCR reaction, U.S. Mosaic Technology company has developed a kind of solid phase PCR system.This system comprises two cover primers, and every cover can be from the target gene both end extension.When primer and DNA sample and PCR reagent mixed mutually, if sample comprises target sequence, DNA just began to synthesize from primer two, and between primer formation double-stranded DNA ring or " bridge ".Because above-mentioned being reflected in the solid phase produces, thereby avoided the primer warfare, and can reduce the residue pollution and repeat initiation.The patent of this solid phase PCR system, protection be simultaneously, be adjacent to be fixed on a pair of primer on the photoconductive fiber end, and with this to the primer amplification target dna, it is final that detect is the bridging DNA that forms between this a pair of primer through amplification.In this technology, if be the wall scroll primer amplification, do not form bridging DNA, then can not detect signal, see (U.S. Pat 5641658, http://www.mosaic.com).
Two. of the present invention specifying:
Gene chip high-flux quick detection technique of the present invention, extending detection technique by solid phase primer (or being called probe) realizes, this technology is that 5 ' end as the oligonucleotide of primer (or being called probe) is connected on the solid phase (being the solid phase carrier of gene chip), after primer and corresponding D NA single-stranded template (being target dna) or RNA (the being target RNA) annealing, use depends on the archaeal dna polymerase of template, in containing the corresponding reaction system of beacon molecule, carry out 5 ' → 3 ' DNA polymerization, 3 ' end along primer extends the strand with target dna strand complementary product D NA, this product D NA strand is labeled the beacon molecule when extending, after extending end, the various free composition of DNA polyreaction is removed in washing.The product D NA strand of beacon molecule firmly is connected on the solid phase on the mark.Detect the beacon signal on the solid phase.If signal is arranged, then representing has target dna or target RNA in the detected sample; If no signal, then representing does not have target dna or target RNA in the detected sample.The specificity of solid phase primer extension detection technique of the present invention is by the length of primer and the annealing temperature and the salt concn decision of primer and target dna strict control in reaction; Sensitivity of the present invention, the strength of signal of promptly mixing beacon then can make more beacon molecule be impregnated in and be exaggerated by the long as far as possible extension of polymerisate dna single chain, and this is first kind of beacon markers method of the present invention.
Second kind of beacon markers method of the present invention comprises two steps, first step is to adopt and above-mentioned first kind of step that the beacon markers method is identical, but do not contain the beacon molecule in the DNA polymerization reaction system, extension obtains not by the polymerisate dna single chain of beacon markers after finishing; Second step is in containing the DNA polymerization reaction system of beacon molecule, carry out the DNA polyreaction second time by polymerisate dna single chain downstream nucleotide sequence complementary therewith, the primer that is not connected on the solid phase, generate and the new dna single chain that is labeled beacon of former polymerisate dna single chain complementary, only otherwise two complementary two strandss of the formed DNA of polymerisate strands before and after this are unwind, just can determine target gene by detecting these beacon signals that are labeled.
The third beacon markers method of the present invention is, described first kind and second kind of beacon markers method are combined, make the polymerisate dna single chain of first kind of described solid phase primer extension of marking method be labeled beacon, and then with second step of described second kind of beacon markers method produce one with it complementary be labeled the strand of beacon, detect the beacon signal of these two formed dna double chains of strand complementary pairing and just can determine target gene.
The employed archaeal dna polymerase that depends on template in the solid phase primer extension of the present invention, comprise, but be not limited to e. coli dna polymerase I (holoenzyme), e. coli dna polymerase I Klenow fragment, T4 phage DNA polysaccharase, Sequenase (T7 phage DNA polysaccharase), Taq enzyme, ThermoScript II.
The solid phase carrier that gene chip of the present invention adopted is selected from any material that can be used for preparing gene chip, and it includes, but not limited to sheet glass, silicon chip and plastic sheet.Described solid phase carrier after different chemically modifieds, have on its surface can immobilized nucleic acid molecule active group.These groups include, but not limited to amino (NH
2), aldehyde radical (CHO), carboxyl (COOH) and sulfydryl (SH).
Gene chip of the present invention is the specific nucleic acid primer (or being called probe) that is connected with on described solid phase carrier at specific target gene or target dna or target RNA, described primer is a single strand dna, its nucleotide sequence is selected from arbitrary chain in target gene or the dna double chain or is selected from the complementary sequence of target RNA, and one of the strand after can unwinding with this target gene or DNA under suitable condition or with target RNA specific hybridization.Described primer can be by DNA automatically synthetic or pcr amplification obtain, its length is at least 15 Nucleotide, preferably be at least 20 Nucleotide, more preferably be at least 30 Nucleotide, also more preferably be at least 40 Nucleotide, also more preferably be at least 50 Nucleotide, most preferably be at least 60 Nucleotide, also most preferably be at least 70 Nucleotide.Described primer (or being called probe), its 5 ' end can directly be connected on the solid phase carrier, or can optionally add a purpose at its 5 ' end and be to increase the connecting arm that the spatial activity of this primer is beneficial to hybridize, primer is connected on the solid phase carrier by connecting arm.Described connecting arm includes, but not limited to poly thymidine poly (dT) n (n 〉=5), poly uridine poly (dU) n (n 〉=5), poly adenosine poly (dA) n (n 〉=5) or the isometric catenate molecule of polyoxyethylene glycol.
In gene chip of the present invention, 5 ' of described nucleic acid primer is held or is held 5 ' end of the connecting arm that links to each other by modification (connection) active group to be arranged with 5 ' of primer.These active groups include, but not limited to carboxyl (COOH), amino (NH
2), sulfydryl (SH), phenylo boric acid (phenylboronic acid), vinyl (CH3C=CH2) etc.As 5 ' end of primer or with 5 ' end of the 5 ' connecting arm that links to each other of end of primer by carboxyl modified, then the amino of carboxyl and surface of solid phase carriers reacts, formation amido bond and this primer is connected on the solid phase carrier with covalent linkage.As 5 ' end of primer or with 5 ' end of the 5 ' connecting arm that links to each other of end of primer by amido modified, the aldehyde radical or the carboxyl reaction of then amino and surface of solid phase carriers, formation amido bond and this primer is connected on the solid phase carrier with covalent linkage.As 5 ' end of primer or with 5 ' end of the 5 ' connecting arm that links to each other of end of primer by sulfydryl modification, then the sulfydryl of sulfydryl and surface of solid phase carriers reacts the formation disulfide linkage and this primer is connected on the solid phase carrier with covalent linkage.Hold 5 ' end of the connecting arm that links to each other to be modified by the phenylo boric acid base as 5 ' end of primer or with 5 ' of primer, then the salicylhydroxamic acid of phenylo boric acid and surface of solid phase carriers (salicylhydroxamic acid) reaction forms covalent linkage and this primer is connected on the solid phase carrier.As 5 ' end of primer or with 5 ' end of the 5 ' connecting arm that links to each other of end of primer by modified by vinyl, then the sulfydryl of vinyl and surface of solid phase carriers reacts the formation thioether bond and this primer is connected on the solid phase carrier with covalent linkage.Hold 5 ' end of the connecting arm that links to each other to be modified as 5 ' end of primer or with 5 ' of primer by vitamin H (biotin), then vitamin H combines with the Streptavidin (streptavidin) or the avidin (avidin) of surface of solid phase carriers, forms non covalent bond and primer is fixed on the solid phase carrier.
The preparation of gene chip of the present invention, be utilize little ranking method according to the design with described nucleic acid primer (or being called probe) in an orderly manner point sample to solid phase carrier, 5 ' end by nucleic acid primer or react with 5 ' of the 5 ' connecting arm that links to each other of end of primer terminal modified active group and the corresponding active group of carrier surface, form covalently or non-covalently and connect, nucleic acid primer is fixed on the carrier, thereby makes gene chip.Simultaneously, on prepared gene chip, each nucleic acid primer (or probe) all has the fixed position, therefore after they obtain by the solid phase primer extension being labeled the product D NA chain of beacon with target dna strand complementary, by can determining the specific nucleic acid probe that it is corresponding, thereby determine the target gene corresponding with it to the addressing of the beacon signal that gene chip sent.
Solid phase primer extension of the present invention detects extension and the marking method of target dna or RNA, can utilize various nucleic acid known in the art to extend and marking method, the Oligonucleolide primers or the probe that are connected on the gene chip solid phase carrier are extended, produce and target dna or RNA complementary strand, this strand when extending by beacon markers and/or subsequently by another new being labeled strand and strengthening mark or be labeled of complementary with it.Described extension and marking method include, but not limited to methods such as PCR, RT-PCR, in-vitro transcription, random primer labelling, nick translation and terminal metastatic marker.Described beacon molecule can be fluorescein, isotropic substance or vitamin H; Described fluorescein includes, but not limited to Cy3, Cy3.5, Cy5, fluorescein isothiocyanate, texas Red; Described isotropic substance includes, but not limited to
32P,
35S,
3H; Described vitamin H includes, but not limited to vitamin H, photobiotin, long arm photobiotin.Methods such as PCR, RT-PCR, in-vitro transcription, random primer labelling, nick translation and terminal metastatic marker are known in the art.Behind extension and the mark, adopt corresponding detecting method that gene chip is carried out scanning analysis,, judge the target dna or the RNA that are detected according to the location addressing of catching positive signal according to selected beacon molecule.Required all commercializations of equipment in signals collecting and analytic process.
From operation steps, the present invention directly carries out the solid phase primer extension with target dna or RNA on chip, and simple washing can be carried out fluorescent scanning after several minutes was finished to the dozens of minutes reaction, and is fast simple; Avoided commonly used at present, again with the complicated tediously long step that is about 6 to 10 hours consuming time of the probe hybridization on amplified production and the chip earlier with pcr amplification target dna or RNA.Specificity from detected result, the present invention is when carrying out the solid phase primer extension, because different primers all is fixed on different zones, even therefore detect thousands of different target dna or RNA simultaneously, these primers can the phase mutual interference, detects unapproachable high-throughput, high accuracy detection thereby really reached present gene chip; And factor affecting such as the hybridization specificity that determined of the pairing of the base complementrity when being subjected to primer length, primer and template annealing, strict annealing temperature and salt concn, the present invention has high degree of specificity to target dna and the RNA that is detected.From the strength of signal of detected result, because when the primer solid phase was extended, polymerisate dna single chain can be tried one's best to extend also longways and constantly be mixed the beacon molecule, so strength of signal is big, signal combination is firm.
Be described by preparation, solid phase primer extension, detection method and the application thereof of following examples gene chip of the present invention.It should be noted that following embodiment just is used for explanation and unrestricted the present invention, the technology that other detect target dna or target gene by gene chip well-known to those skilled in the art all within the scope of the invention.Embodiment 1 is connected primer by amido linkage and makes gene chip on the solid phase carrier, extends the solid phase primer to detect with the Taq enzyme
Target gene (target dna): detect outside helicobacter pylori (H.pylori) urease B gene and the intestinal bacteria
The gene chip of membranin phoE gene.
1. primer is synthetic: utilize PCR primer-design software Primer Premier5.0 to design the special primer of a helicobacter Pylori urease B gene (ureB): the primer of a 5 ' GTT GCT CCT AAA AAA TCC T 3 ' and an escherichia coli outer membrane protein phoE gene specific: 5 ' AAA GCC GTG GCA CAG GCA AGC GT 3 ', utilize automatic dna synthesizer (PE company) according to the synthetic corresponding Oligonucleolide primers of this dinucleotides primer sequence.In synthetic these two Oligonucleolide primers processes, all add 20 extra thymidines (poly (dT) in 5 ' end of every primer sequence
20), and carry out amido modified to 5 ' end of institute's synthetic oligonucleotide primer thing.By polyvinyl lactam (PAGE) gel electrophoresis the synthetic oligonucleotide is carried out purifying.Described purification process is well known by persons skilled in the art.
2. the preparation of gene chip: two Oligonucleolide primers of synthetic are dissolved in respectively in the aseptic deionized water of proper volume, and the final concentration that makes primer all is 60 μ M.From primer solution, draw 5 μ l respectively and place in the Eppendorf tube, and in Eppendorf tube, add the dimethyl sulfoxide (DMSO) (DMSO) (Sigma, the U.S.) of 5 μ l respectively.Behind primer solution and the abundant mixing of dimethyl sulfoxide (DMSO), get the respective aperture that 5 μ l place 386 orifice plates.With aldehyde group modified slide (CSS-100 Silylated Slides, CEL Associates, Inc., Houston, the U.S.) as solid phase carrier.Utilize GSI Flexys gene chip sample applying instrument (Genomic Solution, the U.S.) with primer according to 3 * 3 rectangular dots lattice point corresponding position (being little ordering point sample) to the slide, the aldehyde radical reaction formation amido bond of the amino of primer 5 ' end and surface of glass slide and primer is fixed on the slide.Like this, the Oligonucleolide primers matrix on the different positions just corresponds respectively to helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene.Under the room temperature gene chip lucifuge was placed 24 hours at least.
3. the Taq enzyme extends solid phase primer detection helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene: according to DNA extraction method well known by persons skilled in the art, prepare four kinds of DNA samples to be measured: the DNA sample that contains the helicobacter Pylori urease B gene, the DNA sample that contains escherichia coli outer membrane protein phoE gene, be mixed with the DNA sample of helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene, the DNA sample that does not contain helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene, utilize above-mentioned gene chip, adopt solid phase primer extension technology to detect this four kinds of DNA samples respectively.
In brief, preparation 20-200 μ l PCR reaction solution, its composition is as follows: 1 * Taq enzyme reaction buffer solution, 2.0mM MgCl
2, the dCTP of dATP, the dTTP of each 200 μ M and dGTP, each 100 μ M and Cy3-dCTP, Taq enzyme 0.5u/20 μ l, an amount of DNA sample to be measured (pre-sex change or pre-sex change all can).Get the PCR reaction solution and join in right amount in the reaction tank on the gene chip, the confined reaction pond is in case the reaction solution evaporation.The PCR reaction of beginning solid phase primer extension.
Reaction tank can be formed by following mode: (1) is around Oligonucleolide primers matrix on the gene chip, with the TR thin rubber bar that scribbles viscose glue, silica gel strip, plastic strip etc., or gene frame original position frame (GENE company) surrounds a reaction tank, after adding the PCR reaction solution, cover glass or plastic sheet or polyethylene film etc. after covering slide or silanization on the reaction tank make reaction tank airtight.(2) Self-seal that adds GENE company in the PCR reaction solution prevents steaming agent automatically, drip the PCR reaction solution then and cover the Oligonucleolide primers matrix, cover glass or plastic sheet or polyethylene film etc. after covering slide or silanization on the PCR reaction solution, in the PCR reaction process, because high temperature, the liquid evaporation at edge and form the solid gums wall, thus an airtight PCR reaction tank formed.(3) in advance the solid phase carrier of gene chip is carried out etching, make in the zone of carrying out little ordering point sample and form depression; After this sunk area carries out little ordering point sample, add the PCR reaction solution, directly the surface of solid phase carriers at gene chip covers cover glass, slide or the plastic sheet etc. that scribble viscose glue, can form airtight reaction tank.(4) in advance the solid phase carrier of gene chip is carried out etching, make in the zone of carrying out little ordering point sample and form depression; After this sunk area carries out little ordering point sample, directly cover cover glass, fragmentation or the plastic sheet etc. that scribble viscose glue at surface of solid phase carriers at gene chip, form reaction tank, this reaction tank respond liquid import and outlet, after adding the PCR reaction solution, import of off-response liquid and outlet promptly form airtight reaction tank.
Also have a kind ofly need not form the mode that the PCR reaction of solid phase primer extension just can be carried out in the confined reaction pond, promptly prepare the PCR reaction solution of comparatively large vol, directly gene chip is immersed and just can carry out PCR in the PCR reaction solution and react at gene chip.
Described PCR reaction is, with 88-94 ℃ of 10-300 second, 40-65 ℃ of 10-300 second, the loop parameter of 72 ℃ of 10-300 seconds carries out carrying out extension 10 seconds to several minutes in 72 ℃ at last more than 1 circulation.
After PCR reaction finishes, plant reaction tank, throw off things such as the cover glass that covers on the reaction tank or plastic sheet, expose little ordering point sample zone for above-mentioned (1), (2), (3); For (4) above-mentioned kind reaction tank, remove the PCR reaction solution from reaction solution outlet or inlet; For above-mentioned gene chip is directly immersed the reaction that the PCR reaction solution is carried out, then directly take out gene chip.(12 * SSC 0.2%SDS) waits little ordering point sample district of washing gene chip, removes the composition of the unreacted PCR reaction solution of chip surface for water or TE damping fluid or hybridization buffer.
Washing finishes, and takes out the natural airing in back.Utilize GeneTAC 2000 gene chip scanning instruments (Genomic Solution Inc., the U.S.), under the excitation wavelength of 532nm, scan and automatic exposure.Analysis scan result, and fluorescent signal carried out addressing, and corresponding with specific oligonucleotide probe, determine target dna or target gene in the sample.
Detected result: 1. for the sample that only contains the helicobacter Pylori urease B gene DNA, gene chip only sends fluorescent signal in the matrix point sample zone of helicobacter Pylori urease B gene specific primer.2. for the sample that only contains escherichia coli outer membrane protein phoE gene DNA, gene chip only sends fluorescent signal in the matrix point sample zone of escherichia coli outer membrane protein phoE gene specific primer.3. for the sample that is mixed with helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene DNA, gene chip has all sent fluorescent signal in the matrix point sample zone of helicobacter Pylori urease B gene specific primer and the matrix point sample zone of escherichia coli outer membrane protein phoE gene specific primer.4. for the sample that does not contain helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene DNA, no fluorescent signal occurs on the gene chip.Embodiment 2 is connected primer by disulfide linkage and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with the Taq enzyme: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.
1. primer is synthetic: the nucleotide sequence of institute's synthetic primer is with embodiment 1, all adds 20 in 5 ' end of every primer sequence
Individual extra uridine (poly (dU)
20), but what carry out is that sulfydryl is repaiied to 5 ' end of institute's synthetic oligonucleotide primer thing
Decorations, particularly, the group of modification is the C with Cruachem company
6Disulphide phosphoramidite connects
5 ' the end of receiving primer obtains.
2. the preparation of gene chip: method and step are with embodiment 1, but used solid phase carrier is to use 3-Mercaptopropyl
Trimethoxysilane handles and the slide of the sulfydryl modification that obtains.The C of the sulfydryl of surface of glass slide and primer 5 ' end
6
Disulphide phosphoramidite reaction forms disulfide linkage and primer is fixed on the slide.
3. extend the solid phase primer with the Taq enzyme and detect helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene:
Washing and the scanning and the inspection of the PCR reaction conditions of solid phase primer extension, the preparation of reaction tank, reaction back gene chip
Survey the result all with embodiment 1.Embodiment 3 is connected primer by disulfide linkage and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with e. coli dna polymerase I Klenow fragment: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.1. remove in 5 ' end of every primer sequence and all add 20 extra adenosines (poly (dA)
20) outside, primer sequence, to the preparation of the 5 ' sulfydryl modification that carries out of end of institute's synthetic oligonucleotide and gene chip all with embodiment 2.2. e. coli dna polymerase I Klenow fragment is extended the solid phase primer to detect helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene:
According to four kinds of DNA samples to be measured of embodiment 1 described method preparation,, promptly place ice bath after 2 minutes immediately 99 ℃ of sex change with they pre-sex change.Utilize e. coli dna polymerase Klenow fragment according to the primer on the method known to those skilled in the art extension gene chip and carry out fluorescein-labelled to extension products dna single chain.Specific as follows: the preparation reaction solution makes DNA sample to be measured after the dCTP of dATP, the dTTP of the e. coli dna polymerase Klenow fragment that contains 1 * e. coli dna polymerase Klenow fragment reaction buffer, 50 units in 50 μ l final volume, each 200 μ M and dGTP, each 100 μ M and Cy3-dCTP and an amount of pre-sex change.With Oligonucleolide primers matrix area on the reaction solution covering gene chip, in 37 ℃ of incubations 2 hours.After reaction finished, (12 * SSC 0.2%SDS) waited little ordering point sample district of washing gene chip, removes the unreacted reaction solution composition of chip surface for water or TE damping fluid or hybridization buffer.
Washing finishes, and takes out the natural airing in back.Utilize GeneTAC 2000 gene chip scanning instruments (Genomic Solution Inc., the U.S.), under the excitation wavelength of 532nm, scan and automatic exposure.Analysis scan result, and fluorescent signal carried out addressing, and corresponding with the specific oligonucleotide primer, determine target dna or target gene in the sample.
Detected result: with embodiment 1.Embodiment 4 is connected primer by disulfide linkage and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with e. coli dna polymerase I holoenzyme: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.
Remove in 5 ' end of every primer sequence and all add polyoxyethylene glycol arm (spacer amidite, 18 atom spacerphosphoramidite, Cruachem company) and be equivalent to the polyoxyethylene glycol arm end C that primer 5 ' is held
6Disulphidephosphoramidite carries out outside the sulfydryl modification, and the preparation of the preparation of primer sequence, gene chip, four kinds of DNA samples to be measured and pre-sex change are all identical with embodiment 3.
Utilize e. coli dna polymerase I holoenzyme, according to the primer on the method known to those skilled in the art extension gene chip and carry out fluorescein-labelled to extension products dna single chain.Specific as follows: the preparation reaction solution makes DNA sample to be measured after the dCTP of dATP, the dTTP of the e. coli dna polymerase I holoenzyme that contains 1 * e. coli dna polymerase I reaction buffer, 50 units in 50 μ l final volume, each 200 μ M and dGTP, each 100 μ M and Cy3-dCTP and an amount of pre-sex change.With Oligonucleolide primers matrix area on the reaction solution covering gene chip, in 37 ℃ of incubations 2 hours.
Reacted washing, fluorescent scanning and detected result are with embodiment 3.Embodiment 5 is connected primer by disulfide linkage and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with T4 phage DNA polysaccharase: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.
Synthetic, the preparation of gene chip of primer sequence, primer, the preparation and the pre-sex change of four kinds of DNA samples to be measured are all identical with embodiment 3.
Utilize T4 phage DNA polysaccharase, according to the primer on the method known to those skilled in the art extension gene chip and carry out fluorescein-labelled to extension products dna single chain.Specific as follows: the preparation reaction solution makes DNA sample to be measured after the dCTP of dATP, the dTTP of the T4 phage DNA polysaccharase that contains 1 * T4 phage DNA polymeric enzyme reaction damping fluid, 4 units in 50 μ l final volume, each 200 μ M and dGTP, each 100 μ M and Cy3-dCTP and an amount of pre-sex change.With Oligonucleolide primers matrix area on the reaction solution covering gene chip, in 37 ℃ of incubations 2 hours.
Reacted washing, fluorescent scanning and detected result are with embodiment 3.Embodiment 6 is connected primer by disulfide linkage and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with T7 phage DNA polysaccharase: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.
Synthetic, the preparation of gene chip of primer sequence, primer, the preparation and the pre-sex change of four kinds of DNA samples to be measured are all identical with embodiment 3.
Utilize T7 phage DNA polysaccharase, according to the primer on the method known to those skilled in the art extension gene chip and carry out fluorescein-labelled to extension products dna single chain.Specific as follows: the preparation reaction solution makes DNA sample to be measured after the dCTP of dATP, the dTTP of the T7 phage DNA polysaccharase that contains 1 * T7 phage DNA polymeric enzyme reaction damping fluid, 4 units in 50 μ l final volume, each 200 μ M and dGTP, each 100 μ M and Cy3-dCTP and an amount of pre-sex change.With Oligonucleolide primers matrix area on the reaction solution covering gene chip, in 37 ℃ of incubations 2 hours.
Reacted washing, fluorescent scanning and detected result are with embodiment 3.Embodiment 7 is connected primer by disulfide linkage and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with Sequenase: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.
Sequenase is to have removed 3 ' → 5 ' exonuclease activity through modifying, but the T7 phage DNA polysaccharase that polymerase activity still keeps.
The primer sequence of present embodiment, primer are synthetic, the preparation and the pre-sex change of the preparation of gene chip, four kinds of DNA samples to be measured are all identical with embodiment 3.
Utilize T7 phage DNA polysaccharase, according to the primer on the method known to those skilled in the art extension gene chip and carry out fluorescein-labelled to extension products dna single chain.Specific as follows: the preparation reaction solution makes DNA sample to be measured after the dCTP of dATP, the dTTP of the Sequenase that contains 1 * Sequenase reaction buffer, 4 units in 50 μ l final volume, each 200 μ M and dGTP, each 100 μ M and Cy3-dCTP and an amount of pre-sex change.With Oligonucleolide primers matrix area on the reaction solution covering gene chip, in 37 ℃ of incubations 2 hours.
Reacted washing, fluorescent scanning and detected result are with embodiment 3.Embodiment 8 is connected primer by disulfide linkage and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with fowl source ThermoScript II: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.
The primer sequence of present embodiment, primer are synthetic, the preparation and the pre-sex change of the preparation of gene chip, four kinds of DNA samples to be measured are all identical with embodiment 3.
Utilize fowl source ThermoScript II, according to the primer on the method known to those skilled in the art extension gene chip and carry out fluorescein-labelled to extension products dna single chain.Specific as follows: the preparation reaction solution makes DNA sample to be measured after the dCTP of dATP, the dTTP of the fowl source ThermoScript II that contains 1 * fowl source ThermoScript II reaction buffer, 4 units in 50 μ l final volume, each 400 μ M and dGTP, each 200 μ M and Cy3-dCTP and an amount of pre-sex change.With Oligonucleolide primers matrix area on the reaction solution covering gene chip, in 42 ℃ of incubations 2 hours.
Reacted washing, fluorescent scanning and detected result are with embodiment 3.Embodiment 9 is connected primer by disulfide linkage and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with mouse source ThermoScript II: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.
The primer sequence of present embodiment, primer are synthetic, the preparation and the pre-sex change of the preparation of gene chip, four kinds of DNA samples to be measured are all identical with embodiment 3.
Utilize mouse source ThermoScript II, according to the primer on the method known to those skilled in the art extension gene chip and carry out fluorescein-labelled to extension products dna single chain.Specific as follows: the preparation reaction solution makes DNA sample to be measured after the dCTP of dATP, the dTTP of the mouse source ThermoScript II that contains 1 * mouse source ThermoScript II reaction buffer, 4 units in 50 μ l final volume, each 400 μ M and dGTP, each 200 μ M and Cy3-dCTP and an amount of pre-sex change.With Oligonucleolide primers matrix area on the reaction solution covering gene chip, in 37 ℃ of incubations 2 hours.
Reacted washing, fluorescent scanning and detected result are with embodiment 3.Embodiment 10. is connected primer by thioether bond and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with the Taq enzyme: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.1. primer is synthetic: the nucleotide sequence of institute's synthetic primer is with embodiment 1, what carry out is modified by vinyl but to 5 ' end of primer, particularly, the group of modification obtains the 5 ' end that the Acrydite of Mosaic Technologies company is connected to primer.2. the preparation of gene chip: the treatment process of solid phase carrier is with embodiment 2, is to handle and the slide of the sulfydryl modification that obtains with 3-Mercaptopropyl trimethoxysilane.The vinyl reaction of the sulfydryl of surface of glass slide and primer 5 ' end forms thioether bond and primer is fixed on the slide.3. extend the solid phase primer with the Taq enzyme and detect helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene: the washing of the PCR reaction conditions of solid phase primer extension, the preparation of reaction tank, reaction back gene chip and scanning and detected result are all with embodiment 1.Embodiment 11. is connected primer by non covalent bond and makes gene chip on the solid phase carrier, carries out the solid phase primer extension to detect target gene (target dna) with the Taq enzyme: the gene chip that detects helicobacter pylori (H.pylori) urease B gene and escherichia coli outer membrane protein phoE gene.1. primer is synthetic: the nucleotide sequence of institute's synthetic primer is with embodiment 1, but when synthetic with vitamin H (biotin) on 5 ' the end mark of primer.4. the preparation of gene chip: with Streptavidin (streptavidin) or avidin (avidin) the concentration bag quilt with 5mg/L, 4 ℃ are spent the night with solid phase carrier.Add encapsulant then, 4 ℃ are spent the night.Wash the back seasoning with water, put 4 ℃ of preservations, thereby obtain the slide of Streptavidin or avidin modification.The vitamin H of the Streptavidin of surface of glass slide or avidin and primer 5 ' end combines, and forms non covalent bond and primer is fixed on the slide.5. extend the solid phase primer with the Taq enzyme and detect helicobacter Pylori urease B gene and escherichia coli outer membrane protein phoE gene: the washing of the PCR reaction conditions of solid phase primer extension, the preparation of reaction tank, reaction back gene chip and scanning and detected result are all with embodiment 1.
Claims (5)
1. the gene chip solid phase primer extension technology that whether has target dna or RNA in the test sample, it is characterized in that: 1. described gene chip is made for according to certain ordering 5 ' of oligonucleotide being held on the solid phase carrier that is connected gene chip, and it is to be connected on the solid phase carrier of gene chip by covalent linkage or non covalent bond that 5 ' of described oligonucleotide is held; 2. described solid phase primer is the oligonucleotide that described 5 ' end is connected with the solid phase carrier of gene chip by covalent linkage or non covalent bond, one of two dna single chains that the nucleotide sequence of 3 ' end regions of described solid phase primer can be produced through sex change (or unwinding) with target dna are annealed because of complementary pairing, or can anneal because of complementary pairing with target RNA; 3. after described solid phase primer extension is described solid phase primer and described target dna strand or target RNA annealing, under the archaeal dna polymerase effect that depends on template, along primer 3 ' end extend and target dna strand or target RNA complementary product D NA strand.4. described product D NA strand is when extending or be labeled the beacon molecule subsequently, by the signal of beacon molecule on the detection gene chip, thereby judges whether have described target dna or target RNA in the sample.If the signal of beacon molecule is arranged, target dna or target RNA are then arranged, in the sample if the signal of no beacon molecule the solid phase primer extension does not then take place, driftlessness DNA or target RNA in the sample.
2. the described archaeal dna polymerase that depends on template of claim 1 is: e. coli dna polymerase I (holoenzyme), e. coli dna polymerase I Klenow fragment, T4 phage DNA polysaccharase, Sequenase (T7 phage DNA polysaccharase), Taq enzyme, ThermoScript II.
3. the described beacon molecule of claim 1 is fluorescein, isotropic substance or vitamin H.
4. the described solid phase primer of claim 1, can add spatial activity that a purpose is to increase this primer at its 5 ' end that is connected with gene chip solid phase carrier and be beneficial to the connecting arm of annealing or hybridizing, described connecting arm is: poly thymidine poly (dT) n (n 〉=5), poly uridine poly (dU) n (n 〉=5), poly adenosine poly (dA) n (n 〉=5) or the isometric catenate molecule of polyoxyethylene glycol.
5. the described solid phase primer extension of claim 1 The Application of Technology.
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| CN 01123528 CN1400315A (en) | 2001-07-30 | 2001-07-30 | Gene chip high-flux quick detection technique |
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| CN 01123528 CN1400315A (en) | 2001-07-30 | 2001-07-30 | Gene chip high-flux quick detection technique |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005001123A1 (en) * | 2003-06-30 | 2005-01-06 | Tsinghua University | Dna chip based genetic typing |
| CN102321622A (en) * | 2011-08-04 | 2012-01-18 | 盛司潼 | Primer-solid-phase carrier composite and methods for preparing primer-solid-phase carrier and sequencing |
| CN106011238A (en) * | 2009-03-06 | 2016-10-12 | 弗赖堡阿尔伯特-路德维格大学 | Device and method for producing a replicate or derivative from an array of molecules, and applications thereof |
-
2001
- 2001-07-30 CN CN 01123528 patent/CN1400315A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005001123A1 (en) * | 2003-06-30 | 2005-01-06 | Tsinghua University | Dna chip based genetic typing |
| US7718362B2 (en) | 2003-06-30 | 2010-05-18 | Capitalbio Corporation | DNA chip based genetic typing |
| CN106011238A (en) * | 2009-03-06 | 2016-10-12 | 弗赖堡阿尔伯特-路德维格大学 | Device and method for producing a replicate or derivative from an array of molecules, and applications thereof |
| CN102321622A (en) * | 2011-08-04 | 2012-01-18 | 盛司潼 | Primer-solid-phase carrier composite and methods for preparing primer-solid-phase carrier and sequencing |
| CN102321622B (en) * | 2011-08-04 | 2016-03-30 | 盛司潼 | A kind of primer-solid-phase carrier composite and preparation thereof and the method for checking order |
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