CN1733933A

CN1733933A - Exonuclease III digesting Label-dsDNA microarray chip for detecting transcription factor protein

Info

Publication number: CN1733933A
Application number: CN 200410041683
Authority: CN
Inventors: 王进科
Original assignee: Nanjing Xinyihua Group Co Ltd
Current assignee: Nanjing Xinyihua Group Co Ltd
Priority date: 2004-08-13
Filing date: 2004-08-13
Publication date: 2006-02-15

Abstract

Disclosed is a transcription factor protein detection method through exonuclease III digestion Label-dsDNA micro array chips, wherein the expression and activation levels of the transcription factor are analyzed through the following steps: (1) preparing Label-dsDNA micro array chips, (2) reacting the transcription factor with Label-dsDNA micro array chips, (2) reacting the exonuclease III with Label-dsDNA micro array chips, (1) proceeding detection and analysis to the Label-dsDNA micro array chips.

Description

Exonuclease III digestion Label-dsDNA micro-array chip detects transcription factor protein

One, technical field:

Transcription factor (transcription factor) is the key protein that a class is responsible for gene expression regulation (geneexpression regulation) in the bioprotein group (proteomics), being the hinge of gene expression regulation path (pathway) and network (network), is the important object of functional genome (functional genomics) and proteome research; All there is confidential relation in numerous disease with the unconventionality expression (expression) of transcription factor and activation (activation), becomes the important target spot (drug target) of transcribing treatment (transcriptional therapy) and drug research.The check and analysis of transcription factor expression and activation degree are the main means of its function of research.This patent has proposed a kind of novel method that detects transcription factor expression and activation levels, this method will be for being that transcription factor correlative study in molecular biology (molecular biology), functional genomics, proteomics and biomedicine (Biomedicine) field provides a kind of new check and analysis technology, can promote the scientific research that transcription factor in these fields is relevant, and a kind of diagnostic techniques of disease-related transcription factor and drug screening (drug screening) technology that transcription factor is target spot are provided in biomedical sector.

Two, background technology:

Transcription factor protein is the key protein that a class is responsible for gene expression regulation in the bioprotein group, is the hinge of gene expression regulation path and network, is the important object of functional genome and proteome research; All there is confidential relation in numerous disease with the unconventionality expression and the activation of transcription factor, becomes the important target spot of transcribing treatment and drug research.The check and analysis of transcription factor expression and activation degree are the main means of its function of research.Therefore, related detection analysis transcription factor expression and activation degree Study on Technology are subjected to the attention of scientific circles always.

Molecular biology research shows, context (context) at gene exists some performances to start (promote), strengthens (enhance) or the specific dna sequence of (attenuate) genetic transcription effect that decays, be called cis-acting elements (cis-actingelements), as promotor (promoter), enhanser (enhancer), attenuator (attenuater); Transcription factor protein is the special protein of a class in the bioprotein group, after finishing its translation (translation) in the tenuigenin of this proteinoid, being subjected to specificity factor at normal cell functional status or cell induces down, enter nucleus, combine with the cis-acting elements generation specific recognition in the genome, constitutive gene transcriptional machinery (transcription apparatus) is realized the adjusting function of its genetic expression being called trans-acting factor (trans-acting factors).Transcription factor protein and the primary link of cis-acting elements constitutive gene transcriptional machinery are that some have the special construction transcription factor and directly combine (sequence-specific binding) with cis-acting elements dna sequence dna generation sequence-specific in the transcription factor protein, finish the first step of genetic transcription machine assembling.Therefore, the technology of check and analysis transcription factor expression and activation degree mainly is based upon this level of DNA/ protein interaction, i.e. it is probe that utilization contains the DNA of cis-acting elements, surveys the expression and the activation of transcription factor.

Up at present, scientists has been set up multiple based on the transcription factor expression of this level of DNA/ protein interaction and the check and analysis technology of activation degree.That wherein the most classical is electrophoretic mobility shift assay (Electrophoresis MobilityShift Assay), i.e. gel shift experiment (gel shift assay).This technology generally is that synthetic contains transcription factor binding sequence (consensus, binding sites) two strands (double-stranded) oligonucleotide (oligonucleotides), and use the labelled with radioisotope oligonucleotide, with labeled oligonucleotide mix with the cell or tissue extract that contains transcription factor protein hatch for some time after, carry out nature polyacrylamide gel electrophoresis (native polyacrylamide gel eletrophoresis, PAGE), separated free (free DNA) and with the DNA (retarded DNA) of protein bound, DNA (retarded DNA) manifesting by X-exographX exposure with protein bound reflects transcription factor expression and activation degree.This technology still is used for the transcription factor protein check and analysis at present very effectively.But there be the defective of radio-labeling to experimenter and environmental hazard.Therefore, this technology had been carried out afterwards nonradioactive labeling's improvement.Promptly use digoxin (digoxigenin, DIG) labeled oligonucleotide, carry out the nature polyacrylamide gel electrophoresis, again electrophoresis product transfer printing (blotting) is arrived on the nylon membrane media such as (nylon membrane), rely on the DIG antibody of alkaline phosphatase or horseradish peroxidase coupling connection and adding lustre to (colorimetric) or luminous (chemiluminescent) substrate (substrate) of corresponding enzyme, carry out that chemistry adds lustre to or luminous detection, reflect transcription factor expression and activation degree.Also there is employing fluorescence (fluorescent) labeled oligonucleotide to carry out the improvement technology of electrophoretic mobility shift assay.These technology also can perform well in the transcription factor protein check and analysis.But still there is the shortcoming of self in these technology, though be the shortcoming that they have avoided classical radiolabeled probe's gel shift experiment, but bring the complicated and more influence factor of experiment flow simultaneously, as the high background of nylon membrane experiment, quote problems such as device requirement and experimental cost raising.Simultaneously, these improvement technology are not fundamentally broken away from the technological thought of kind of gel shift experiment, can't overcome the gel shift experiment big to laboratory sample quantity demand, test defectives such as length consuming time, analysis efficiency are low.

In view of the shortcoming that these technology still exist at present, set up and be different from the transcription factor expression of gel shift experiment fully on the technological thought angle and activation detects and analytical technology is very important.We have carried out many correlative studys for this reason, and have developed some new technology.For example, utilize the strategy of biochip technology, the double-stranded DNA that we once were devoted to contain the transcription factor protein binding site is fixed to solid phase carrier such as surface of glass slide, preparation double-stranded DNA micro-array chip (double-stranded DNA microarray chip) is used for transcription factor expression and activatory and detects and analyze.We have invented technology (the Chinese patent ZL02112780.8 of several preparation double-stranded DNA micro-array chips, 02137945.9,03152881.3), and successfully prepared and be exclusively used in the double-stranded DNA micro-array chip (Chinese patent 03132206.9) that detects transcription factor protein NF-κ B, and set up thus a kind of on " DNA/ protein " interactional molecule aspect from complex component material such as Chinese medicine and combinatorial chemistry mixture high flux screening, catch the technology (Chinese patent 03152882.1) with the separate targets molecule.

The validity of technology and practicality are the lifeblood of technology.Though our double-stranded DNA (dsDNA) micro-array chip technology has reached the practicability level, but we notice that applying of this technology still faces very big difficulty, specifically be reflected in following some: the one, the double-stranded DNA micro-array chip of the method preparation before us is difficult to realization and detects many transcription factors simultaneously, does not reach the purpose of high throughput testing; The 2nd, in detection, need to prepare the antibody of transcription factor protein to be detected; The 3rd, need carry out proteic fluorescent mark.These shortcomings make us must prepare various double-stranded DNA micro-array chips, realize detection to multiple transcription factor, this can not realize that but dna microarray chip high-throughput obtains the function of bioinformation, the preparation of antibody and mark are a kind of expensive work of wasting time and energy in addition, greatly limited the application of chip, the complicacy and the difficulty of the experiment that increases have improved experimental cost.Therefore, set up a kind of Antibody Preparation and mark of not relying on, and can realize really that the double-stranded DNA micro-array chip technology of many transcription factors high throughput testing is in demand.Thus; we put forth effort to improve this technology; dna microarray chip technology and exonuclease III protection analysis (ExoIII Protection Assay) technology are combined, proposed the present invention's " exonuclease III digestion Label-dsDNA micro-array chip detects transcription factor protein " technology.This technology is in the same place the high throughput testing function of double-stranded DNA micro-array chip technology with the advantages of exonuclease III protection analytical technology; numerous and diverse experimental procedures such as Antibody Preparation and fluorescent mark have been broken away from; only need on chip, carry out simple and exonuclease III protection analysis that cost is very cheap; just can realize high throughput testing to a large amount of transcription factor proteins; this improvement; greatly reduce the preparation and the use cost of Label-dsDNA micro-array chip detection transcription factor protein, can solve the Label-dsDNA micro-array chip The Application of Technology problem that we set up well.

Three, summary of the invention:

(1), goal of the invention

The purpose of this invention is to provide a kind of Label-dsDNA of preparation micro-array chip and realize the low-cost novel method that detects multiple transcription factor expression and activation levels of high-throughput; be convenient to utilize the preparation of gene chip; detection technique and equipment; as DNA in sheet original position synthetic technology; DNA chip point sample technology of preparing and gene chip fluorescent scanning analytical technology etc.; prepare the Label-dsDNA micro-array chip of band special marking such as fluorescence molecule; rely on once simple exonuclease III protection to analyze again, just can detect the expression and the activation levels of multiple transcription factor in the solution to be checked.This technology will provide a kind of new experimental technique for the correlative study of the transcription factor in the fields such as molecular biology, functional genomics, proteomics and biomedicine, promote the relevant scientific research of transcription factor in these fields, and study at the clinical detection and the drug screening of transcription factor.

(2), technical scheme

This patent has proposed a kind of new technology that detects transcription factor expression and activation levels, i.e. " exonuclease III digestion Label-dsDNA micro-array chip detects transcription factor protein ".

Use this method detection transcription factor protein expression and activation levels to comprise the steps:

A) prepare the Label-dsDNA micro-array chip;

B) transcription factor protein and Label-dsDNA micro-array chip reaction;

C) exonuclease III and Label-dsDNA micro-array chip reaction;

D) the Label-dsDNA micro-array chip is carried out check and analysis.

The technological thought of above step comprises two parts, at first is to prepare the Label-dsDNA micro-array chip, and next uses the Label-dsDNA micro-array chip to detect transcription factor protein.

The preparation of Label-dsDNA micro-array chip is the first step that realizes the technology of the present invention, it also is a very important step, key issue comprises design and prepares the nucleic acid probe of preparation Label-dsDNA micro-array chip, transcription factor protein in conjunction with the reaction of Label-dsDNA micro-array chip, exonuclease III requirements such as good action to the Label-dsDNA micro-array chip.The nucleic acid probe that is used for preparing the present invention's Label-dsDNA micro-array chip structurally should satisfy following condition:

A) nucleic acid probe of being prepared comprises two single stranded nucleic acid molecule A and B with specific structural features;

B) nucleic acid molecule A and B are the nucleic acid molecule of two base sequence reverse complementals;

C) on the double chain acid molecule that nucleic acid molecule A and B annealing back forms, contain that transcription factor protein can be discerned and bonded nucleotide sequence with it, and the number of transcription factor protein binding sequence is not limited in one;

D) 3 of nucleic acid molecule A ' terminal chemical group according to chip surface has carried out corresponding chemically modified, so that it is affixed to chip surface by chemical reaction;

E) after nucleic acid molecule A and the B annealing, 3 of B ' is terminal recessed with the terminal 3 ' end concordant or B of 5 of A ', so that exonuclease is when acting on the AB mixture, and can be from carrying out property of the 3 ' end degraded B of B;

F) 5 of nucleic acid molecule B ' terminal or close 5 ' end contains the Nucleotide that special marking is modified, and the Nucleotide number that special marking is modified is at least one; When the Nucleotide number of special marking modification increases, help improving the susceptibility of detection;

G) Nucleotide modified of the special marking that contained of nucleic acid molecule B, its special marking is that fluorescein, digoxin, vitamin H etc. can rely on its chemical property to carry out the chemical molecular of check and analysis;

H) the transcription factor protein binding sequence of nucleic acid molecule B is between Nucleotide and 3 ' end that special marking is modified; Promptly form the structure of " 5 ' end---Nucleotide---transcription factor protein binding sequence---3 ' end that special marking is modified ".

The preparation of Label-dsDNA micro-array chip among the present invention; its technological thought derives from the experimental technique that we carry out the conjugated protein affinity chromatography separation and purification of DNA to the medium of utilization DNA bag quilt in double-stranded DNA micro-array chip Study on Technology and invention, the traditional molecular biology, and utilization exonuclease III carries out DNA protection analytical technology researching DNA/protein interaction in the molecular biology.The key problem in technology of preparation Label-dsDNA micro-array chip is how double chain acid molecule to be affixed to chip surface securely, make the fixed double chain acid molecule both kept with liquid phase in transcription factor protein combine actively, can stand washing in the testing process again and unlikely coming off.

Owing to occur between protein and the double-strandednucleic acid the interaction of SDBP (as transcription factor) and its target DNA, therefore the dna probe that is used for detecting transcription factor generally is a double chain acid molecule, the oligonucleotide annealing preparation dna probe of two base sequence reverse complementals of chemosynthesis commonly used.At this moment, on the synthetic oligonucleotide, to add the binding sequence of transcription factor protein, be generally common motif (consensus).The FRET nucleic acid probe for preparing in the present technique is no exception, when design and synthesizing single-stranded nucleic acid molecule A and B, embeds the binding sequence of specific transcription factor protein in the sequence of A, B.For example, when preparation detects the dna probe of transcription factor protein p53, embedding sequence 5 on single stranded nucleic acid molecule A '-AGACATGCCTAGACATGCCT-3 ', and on single stranded nucleic acid molecule B, embed sequence 3 '-TCTGTACGGATCTGTACGGA-5 ', on the nucleic acid probe that A, B annealing back forms, then contain Sequence, this has just constituted the binding sequence of transcription factor protein p53; When preparation detects the dna probe of transcription factor protein SP1 for another example, embedding sequence 5 on single stranded nucleic acid molecule A '-GGGGCGGGGC-3 ', and on single stranded nucleic acid molecule B, embed sequence 3 '-CCCCGCCCCG-5 ', on the nucleic acid probe that A, B annealing back forms, then contain

Sequence has just constituted the binding sequence of transcription factor protein SP1.The binding sequence of transcription factor protein, except that from the natural biology genome, identifying the binding sequence of finding such as common motif (consensus), also can be it to be had the nucleotide sequence of good sequence specificity in conjunction with affinity through the transcription factor protein that experiment in vivo and vitro screens.The binding sequence of transcription factor protein generally should contain flanking sequence (flanking sequence) on the FRET nucleic acid probe of preparation, could be well combine with transcription factor protein identification in the solution, and different transcription factor proteins is also not necessarily identical to the requirement of flanking sequence length, when probe design, should note adding the flanking sequence of useful length at transcription factor protein binding sequence side.

For make the fixed double chain acid molecule have with liquid phase in transcription factor protein combine active, the double chain acid molecule that requirement is used for fixing has enough length, particularly on the double chain acid molecule chimeric transcription factor protein binding sequence, want and chip surface between have sufficient distance, avoid chip surface that nucleic acid molecule is reacted with combination of proteins and cause spatial obstacle (steric hindrance).An end that is used for the nucleic acid molecule that is connected with chip surface should connect long arm molecule (arm, linker, spacer molecules), as C12, PEG (Hexaethylene glycol), etc.Chip surface fixed nucleic acid molecule density (density) also will suit in addition, avoids overstocked nucleic acid molecule to cause the spatial obstacle of protein bound.Since with transcription factor protein generation sequence specific recognition bonded DNA be double-stranded DNA, therefore the fixed nucleic acid molecule should be kept stable double-stranded state in the whole process that detects, avoid the loss of activity of unwinding, two of dna molecular chains should have enough GC content and length for this reason, so that double chain DNA molecule has higher Tm value, certain temperature and salt concn environment in the tolerance testing process and unlikely unwinding.

In order securely double chain acid molecule to be affixed to chip surface, make it can stand washing in the testing process and unlikely coming off, during preparation Label-dsDNA micro-array chip, reply is at solid support such as glass (glass) as chip, silicon chip (silica), gel (gel) etc. carries out surface activation process, physics such as silanization as glass, chemical treatment, make chip surface form specific reactive group (reactive groups), as amino (amino), aldehyde radical (aldehyde), carboxyl (carboxyl), hydroxyl (hydroxyl), thiol group (thiol), N-oxygen succinimide ester (N-oxysuccinimide esters, NOSgroups) etc.These reactive groups can with the respective reaction group generation chemical reaction of chemically modified at the nucleic acid molecule end, form covalent linkage (covalent bond), as schiff bases (Schiff base) etc., with nucleic acid molecule covalently bound fixing (immobilizing) at chip surface.Except this covalently bound, the slide (Streptavidin coated slides) that also can utilize streptavidin bag quilt is fixed to chip surface with the nucleic acid molecule of vitamin H (biotinated) mark.In addition by laying dextran (allyldextran), agarose (agarose), polyacrylamide (polyacrylamide), hydrophilic gel (hydrogel) thin layer (monolayer at chip surface, layer) etc. means bring up reactive group, also can realize the fixing of nucleic acid molecule.

At the fixing double chain acid molecule of chip surface, can realize by number of ways.Generally show as three kinds of modes, the one, at first will be affixed to chip surface with a single stranded nucleic acid molecule of reactive group, by the means of nucleic acid hybridization renaturation another base sequence complementary single stranded nucleic acid molecule is annealed up again, form the fixed double chain acid molecule; The 2nd, earlier with two base sequence complementary single stranded nucleic acid molecules renaturation in liquid phase, form double chain acid molecule, again double chain acid molecule is added on the chip fixedly connected; The 3rd, at first will be affixed to chip surface with a single stranded nucleic acid molecule of reactive group, by the means of nucleic acid hybridization renaturation another base sequence complementary single stranded nucleic acid molecule is annealed up as primer again, by in the DNA of sheet primer extension polyreaction, form the fixed double chain acid molecule at last; In such cases, the Nucleotide that special marking is modified can be arranged in the primer, and when adopting this mode to prepare the Label-dsDNA micro-array chip, can rely on a universal primer and a single stranded DNA chip hybridization that contains the Nucleotide of special marking modification to extend again, reduce preparation cost significantly.First method is difficult to prepare the Label-dsDNA micro-array chip that the present invention proposes, therefore preferably adopt back two kinds of technology to prepare the Label-dsDNA micro-array chip that the present invention proposes, for reducing the chip preparation cost, the third technology is the preferential scheme that adopts of the present invention.

After finishing chip surface and connecting double chain acid molecule, at first to adopt suitable washing measure, remove chip surface is not received the physical adsorption of chip surface by chemical bond-linking nucleic acid molecule, as with 2 * SSC, 0.1% washings washing etc.After washing is removed chip surface and is not received the nucleic acid molecule of chip surface by chemical bond-linking, also to adopt the suitable remaining reactive group of technology deactivation chip surface, avoid them to be in active condition, with protein molecule generation chemical reaction, cause the false positive results of non-specific binding, as adopting NaBH ₄Solution deactivation aldehyde radical, with bovine serum albumin (Bovine serum albumin, BSA) and Tris solution deactivation N-oxygen succinimide ester group etc.

By above-mentioned techniqueflow, then prepared the Label-dsDNA micro-array chip that can be used for the transcription factor protein detection.The quality of chip preparation has determined the Label-dsDNA micro-array chip to detect the reliability of transcription factor protein.Therefore preparing high quality Label-dsDNA micro-array chip is the key point that realizes transcription factor protein measuring ability of the present invention.

Prepared the Label-dsDNA micro-array chip and provide instrument for the detection of transcription factor protein of the present invention.Utilize this Label-dsDNA micro-array chip to detect transcription factor protein, at first to the cell or tissue extract (cell or tissue extracts) of transcription factor protein will be contained, be generally nucleus extract (nuclear extracts), mix with the DNA binding buffer liquid (DNAbinding buffer) of particular chemicals prescription, outside the Label-dsDNA micro-array chip, hatch appropriate time, to hatch thing again is added on the Label-dsDNA micro-array chip, under optimal temperature, continue to hatch appropriate time, transcription factor protein in the extract is combined with Label-dsDNA micro-array chip double-stranded nucleic acid on surface molecule, form " nucleic acid/protein " mixture (complex).It is emphasized that, in this step reaction, should in DNA binding buffer liquid, add an amount of noncompetitive DNA (noncompetitive DNA), as salmon sperm dna (salmon sperm DNA), herring sperm dna (herring spermDNA), carrier DNA[poly (dI-dC), poly (dA-dT)] etc., earlier appropriate time is hatched in " noncompetitive DNA/ extract/binding buffer liquid " system outside the Label-dsDNA micro-array chip, just can be added on the Label-dsDNA micro-array chip, in case form non-specific binding.In addition, before cell or tissue extract and chip are hatched, can carry out sealing treatment with encapsulant to the Label-dsDNA micro-array chip earlier, as BSA, skim-milk, Denhardt reagent, bovine lacto transfer technique optimizer reagent, commercialization Blocking reagent etc., in case form non-specific binding and increase background.The specific reaction of transcription factor protein and Label-dsDNA micro-array chip can be observed its influence to signal and passed judgment on by mix excessive cold dna probe in articulated system.After containing the cell or tissue extract and the reaction of Label-dsDNA micro-array chip of transcription factor protein, remove reaction solution, again with suitable washings washing Label-dsDNA micro-array chip, as contain horse Lay acid buffer (maleate buffer), phosphate buffered saline(PBS) (the phosphate bufferedsaline of micro-nonionic detergent Tween 20, Triton X-100, PBS), to remove the protein of non-specific binding.

After containing the cell or tissue extract and Label-dsDNA micro-array chip incubation reaction and thorough washing of transcription factor protein, exonuclease III reaction solution is added on the Label-dsDNA micro-array chip hatches certain hour, make the DNA on exonuclease III and the Label-dsDNA micro-array chip that endonuclease reaction take place; After endonuclease reaction finishes, remove endonuclease reaction liquid, again with suitable washings washing Label-dsDNA micro-array chip, as contain horse Lay acid buffer (maleate buffer), phosphate buffered saline(PBS) (the phosphate buffered saline of micro-nonionic detergent Tween 20, TritonX-100, PBS), with enzyme of removing non-specific adsorption etc.

The endonuclease reaction of exonuclease III on the Label-dsDNA micro-array chip has two kinds of situations, when the DNA on the Label-dsDNA micro-array chip and transcription factor protein formation " DNA/ transcription factor protein " mixture, then when exonuclease III digests near the DNA that transcription factor protein covers, because the spatial obstacle that transcription factor protein forms, exonuclease III can not move on, then be arranged in the Nucleotide of being modified by digestion chain (nucleic acid molecule B) 5 ' end or near special marking and can not digestedly be released into reaction soln, promptly become the free mononucleotide; And when the DNA on the Label-dsDNA micro-array chip does not form " DNA/ transcription factor protein " mixture with transcription factor protein, then the digestion of exonuclease III can arrive and be digested the Nucleotide that chain (nucleic acid molecule B) 5 ' end or near special marking are modified, then digested being released in the reaction soln of Nucleotide that special marking is modified becomes the free mononucleotide.Endonuclease reaction is removed exonuclease III reaction solution and is washed the Label-dsDNA micro-array chip after finishing, and those are cut the free mononucleotide that is released in the reaction soln by exonuclease III enzyme and then are eliminated from the Label-dsDNA micro-array chip.The content of transcription factor protein is high more in the cell or tissue extract of detected analysis; activity is good more; then when cell or tissue extract and Label-dsDNA micro-array chip are hatched; " DNA/ transcription factor protein " mixture that forms is many more; the Nucleotide that the special marking that do not cut by exonuclease III enzyme by the transcription factor protein protection on the corresponding Label-dsDNA of the being retained in micro-array chip is modified is then many more; detection signal at special marking in the subsequent reactions is then strong more, makes between the detection signal of transcription factor protein and special marking to have the quantity dependence.

Behind exonuclease III reaction end and the thorough washing; will be at the chemical property of special marking on the nucleic acid molecule; carry out subsequent disposal and detection; the present invention is a fluorescent mark to the most direct indicia means of Label-dsDNA probe on the Label-dsDNA micro-array chip; as use FAM; FITC; Cy3; mark dT such as Cy5; like this after chip is with exonuclease III processing and thorough washing; just can directly carry out fluorescent scanning; obtain the variation of fluorescent signal; promptly many more with Label-dsDNA micro-array chip bonded transcription factor protein; the Nucleotide that the special marking of protection on the Label-dsDNA micro-array chip modified is then many more, and fluorescent signal is strong more in then detecting.When the Label-dsDNA probe on the Label-dsDNA micro-array chip adopts other chemical molecular marks, as vitamin H and digoxin commonly used, the Label-dsDNA micro-array chip is after exonuclease III handles, the specificity junction mixture reaction that should on the Label-dsDNA micro-array chip, add fluorescently-labeled special marking, form " special marking/specificity junction mixture-fluorescein " mixture, carry out fluoroscopic examination again.Step " special marking/specificity junction mixture-fluorescein " mixture formation is illustrated to this only to lift two examples herein:

If the Nucleotide that the special marking that contains of nucleic acid molecule B is modified, its special marking are that (digoxigenin in the time of DIG), then adds the specificity junction mixture that the Label-dsDNA micro-array chip hatches and can be fluorescein-labeled DigiTAb digoxin; If the Nucleotide that the special marking that nucleic acid molecule B contains is modified, when its special marking is vitamin H (biotin), then adds the specificity junction mixture that the Label-dsDNA micro-array chip hatches and can be fluorescein-labeled biotin antibody, fluorescein-labeled affinity element (avidin), fluorescein-labeled streptavidin (streptavidin).

Need to prove that every chip that is used to detect all will detect the fluorescent signal that obtains after the end and compare with detecting preceding fluorescent signal with identical instrument record fluorescent signal, could reflect the information of transcription factor protein before detection reaction.

(3), technique effect

Transcription factor protein becomes a class key protein of present genome and the attention of protein groups institute because of the regulation and control of being responsible for genetic expression, and the close relation that exists between numerous disease and transcription factor unconventionality expression and activation, caused the concern of biomedicine field to transcription factor research, transcription factor has become the important target spot of transcribing treatment and drug research.Under these backgrounds, the technical study that relevant functional transcription factor detects and analyzes is subjected to the attention of scientific circles.Multiple transcription factor check and analysis technology based on this level of DNA/ protein interaction is developed, as electrophoretic mobility shift assay.Though this technology and relevant improvement technology are used for the check and analysis of transcription factor at present effectively, they exist radio-labeling to the defective of experimenter and environmental hazard, big to laboratory sample quantity demand, test length consuming time, analysis efficiency is low and is difficult to high pass and quantize to obtain defectives such as biology.To be different from the transcription factor expression of gel shift experiment fully and to activate detection and analytical technology in order to set up on the technological thought angle, we have carried out many correlative studys, and have developed some new technology.Wherein, the most important thing is to utilize the strategy of biochip technology, the double-stranded DNA that will contain the transcription factor protein binding site is fixed to solid phase carrier such as surface of glass slide, preparation double-stranded DNA micro-array chip, be used for transcription factor expression and activatory and detect and analyze (Chinese patent ZL02112780.8,02137945.9,03152881.3,03132206.9), and these technology are used for from complex component material such as Chinese medicine and combinatorial chemistry mixture high flux screening, catch and separate targets molecule (Chinese patent 03152882.1).

But we notice that present the applying of these technology still faces very big difficulty, when particularly using existing Label-dsDNA micro-array chip, be difficult to realize the high throughput testing of many transcription factor proteins, and expensive and loaded down with trivial details material such as the preparation of transcription factor protein antibody and mark is prepared and experimental implementation, has greatly limited the commercialization of Label-dsDNA micro-array chip technology.Therefore; the technology that we adopt the present invention to propose is improved dsDNA micro-array chip technology; be about to dna microarray chip technology and exonuclease III protection analytical technology and combine, proposed " exonuclease III digestion Label-dsDNA micro-array chip detects transcription factor protein " technology.This technology is incorporated into exonuclease III protection analytical technology in the dsDNA micro-array chip technology, avoided in the other technologies dependence to transcription factor protein antibody, and reaches simultaneously the high throughput testing purpose to many transcription factor proteins well.This improvement has really realized the high throughput analysis function of dna microarray chip technology, but has greatly reduced preparation and use cost, can solve the dsDNA micro-array chip The Application of Technology problem that we set up well.

The invention provides the lower detection transcription factor expression of a kind of preparation and use cost and the novel method of activation levels; be convenient to preparation dsDNA micro-array chip under general gene chip appointed condition, rely on traditional exonuclease III protection analytical technology to realize the high throughput testing analysis of many transcription factor expression and activation levels.Our research and research and development of products description of test, under experimental installation conditions such as general gene chip sample applying preparation and fluorescent scanning instrument, use less capital consumption, just can realize the production of Label-dsDNA micro-array chip, go out output and can put into the Label-dsDNA micro-array chip reagent kit that scientific research, medical or similar products application places can be used.Quality examination and analysis revealed, Label-dsDNA micro-array chip reagent kit can reliablely and stablely be used for realizing at short notice to many transcription factor proteins expression, activation and with the interactional check and analysis of DNA.Therefore, this technology will provide a kind of new experimental technique for the correlative study of the transcription factor in the fields such as molecular biology, functional genomics, proteomics, promote the relevant scientific research of transcription factor in these fields.

Of particular note, the Label-dsDNA micro-array chip reagent kit that the technology of the present invention is produced will the drug screening research at transcription factor have very important using value in clinical assistant diagnosis and biomedicine.For example, the expression of transcription factor p53, NF-kB and activation are unusual, and therefore the vital role of bringing into play in numerous disease becomes the important target spot of observing in the clinical diagnosis, also is simultaneously to transcribe treatment and the important target spot of drug screening.We have set about declaring the medicine card of this product at present and have set up the medicine screening system of transcribing of system, promote the commercial applications of product.

Four, description of drawings:

Fig. 1 Label-dsDNA micro-array chip synoptic diagram

Fig. 2 exonuclease III digestion Label-dsDNA micro-array chip detects transcription factor experiment flow legend a

Explain: PBS: protein binding site (Protein Binding Site)

F: fluorescein (Fluorescein)

PB: protein bound (Protein Binding)

EB: exonuclease III is in conjunction with (ExonucleaseIII Binding)

ED: exonuclease III degrade (ExonucleaseIII degrading)

W: washing (Washing)

S: scanning (Scanning)

Fig. 3 exonuclease III digestion Label-dsDNA micro-array chip detects transcription factor experiment flow legend b

Explain: PBS: protein binding site (Protein Binding Site)

D: digoxin (Digoxigenin)

PB: protein bound (Protein Binding)

EB: exonuclease III is in conjunction with (ExonucleaseIII Binding)

ED: exonuclease III degrade (ExonucleaseIII degrading)

FAB: fluorescently-labeled antibodies (Fluorescein-labeled Antibody Binding)

W: washing (Washing)

S: scanning (Scanning)

Fig. 4 exonuclease III digestion Label-dsDNA micro-array chip detects transcription factor experimental result exemplary plot

Explain: the Label-dsDNA micro-array chip of A:FITC mark

Among the B:A Label-dsDNA micro-array chip with handle with ExonucleaseIII again after NF-κ B combines

The Label-dsDNA micro-array chip is handled with ExonucleaseIII after washing with high level salt solution again among the C:B

Five, embodiment

Herein only with Label-dsDNA micro-array chip for preparing the DIG mark and the experiment that detects transcription factor protein NF-κ B, the embodiment of example explanation patent of the present invention.

1, the design of Label-dsDNA probe molecule and chemosynthesis

NF-κ B (Nuclear Factor kappaB) is a class sequence-specific transcription factor, be subjected to multiple material incentives such as inflammatory mediator, virus infection, oxidative stress when cell after, NF-κ B is activated in kytoplasm, enter nucleus and combine with genetic enhancer sequences such as virus, cytokine, somatomedin, cell adhesion molecule, acute phase reaction albumen, enzymes, enhancing gene is transcribed; Thereby in the pathogenic process of a series of diseases that participate in by cytokine, inflammatory mediator and protease, play a significant role.Studies show that in a large number there is very confidential relation in pathologic processes such as NF-κ B overactivity and inflammation, vascular disease, tumour, virus infection, cerebrovascular disease, alzheimer's disease, Parkinson's disease, supersensitivity encephalitis, septic shock, rheumatic arthritis, bronchial asthma, atherosclerosis, ulcerative colitis.At present, NF-κ B has become the important target spot of new drug development, and many biologies and biochemical restrainer can be blocked NF-κ B signal path or suppress NF-κ B/DNA combination, thereby helps the treatment of NF-κ B relative disease.

NF-κ B is a protein family that is made of RelA/p65, RelB, c-Rel, NF-κ B1/p50 and five kinds of albumen of NF-κ B2/p52, can form homodimer (homodimer) or heterodimer (heterodimer) between five kinds of albumen, combine the expression of the regulation and control modern pronunciation of Chinese characters with common motif (consensus) the dna sequence dna generation sequence specific recognition in the genomic dna.Express with the common motif dna sequence dna of NF-κ B bonded in the genome and be generally 5 '-GGGACTTTCC-3 '.Therefore, when design and preparation are used to detect the Label-dsDNA micro-array chip of transcription factor NF-KB, must contain the common motif dna sequence dna of NF-κ B bonded on the Label-dsDNA probe molecule of being prepared, herein we use modal 5 '-GGGACTTTCC-3 ' site.

We the design and by (the BIOASIA Biologic Technology Co.LTD. of Chinese Shanghai Bo Ya Bioisystech Co., Ltd, Shanghai, China) oligonucleotide of synthetic following two base sequence reverse complementals is used to prepare the Label-dsDNA micro-array chip:

5′...TGCGATTAGAACTGGGGACTTTCCCAGCGATTGACTGAGTGGTCGTCGGCGTGTGCTTTT-NH ₂...3′

Wherein

Be dT-DIG.

Oligonucleotide is dissolved in the water with 100 μ M concentration; The mole such as oligonucleotide solution (molar) that will match mixes, and 95 ℃ of insulations 10 minutes slowly are cooled to 15～25 ℃, become the Label-dsDNA probe.With carbonic acid buffer (0.1M Na ₂CO ₃-NaHCO ₃, pH9.0) partial L abel-dsDNA probe dilution being become concentration is 20 μ M spotting solutions, 4 ℃ of preservations are standby.

2, Label-dsDNA micro-array chip preparation

We select the aldehyde group modified slide preparation Label-dsDNA micro-array chip for preparing for use ourselves.Between the aldehyde radical (aldehyde) of last alpha-amino group (primary amino) of DNA this moment and surface of glass slide chemical reaction can take place, form Schiffbase and (C=N-), DNA is fixed on the slide.

In the concrete operations, 20 μ M spotting solutions are put on the aldehyde group modified slide with the gene chip sample applying instrument.Humidity is to hatch 4 hours under 80% the room temperature.Wash slide with water 2 times, remove the not DNA of lotus root connection (coupled).On chip, add 3%BSA solution, hatched 30 minutes for 37 ℃, use the 0.01M PBS solution (pH7.4) of 0.3%Tween 20 to wash chip 2 times again.The purpose that this step handles is to seal the unreacted active group of chip surface.So far, then prepared the Label-dsDNA micro-array chip that can be used for detecting transcription factor NF-KB.Ready-made chip should airtightly be kept at 4 ℃ of refrigerators.Face with preceding at every turn and scan chip, the record fluorescent signal with gene chip scanning instrument.

3, detect transcription factor NF-KB (pure protein) with the Label-dsDNA micro-array chip

With transcription factor NF-KB albumen [κ hNF-κ B (p50), E3770, Promega)] with different concentration dilutions at DNA binding buffer liquid (10mM HEPES pH7.9,50mM KCl, 2.5mM DTT, 0.1mM EDTA, 0.05%NP-40,10%Glycerol, 5% fetal bovine serum) in, 37 ℃ hatch 10 minutes after, be added on the Label-dsDNA micro-array chip with the volume of every array 10 μ l, cover solution with cover glass, 37 ℃ were continued to hatch 50 minutes.Carefully rinse out cover glass with 0.01M PBS solution (pH7.4), wash chip 2 times with the 0.01M PBS solution (pH7.4) that contains 0.3%Tween 20, each 10 minutes.

With exonuclease III (Fermentas Life Science) reaction solution (1U/ μ l Exonuclease III, 66mM Tris-HCl, 30 ℃ of pH8.0at, 0.66mM MgCl ₂) be added on the Label-dsDNA micro-array chip with the volume of every array 10 μ l, cover solution with cover glass, hatched 10 minutes for 37 ℃.(pH7.4) carefully rinses out cover glass with 0.01MPBS solution, washs chip 2 times with the 0.01M PBS solution (pH7.4) that contains 0.3%Tween20, each 10 minutes.

With Cy3 (FluoroLinkTM Cy3 monofunctional dye 5-pack, Cat.No.PA23001, amershampharmacia biotech) the DIG antibody (Anti-Digoxigenin of mark, Fab fragments, Cat.No.1214667, Roche) with 1: 10000 concentration dilution 1 * blocking solution (Cat.No.1096176, Roche) in, volume with every array 10 μ l is added on the Label-dsDNA micro-array chip, hatches 30 minutes for 37 ℃.Carefully rinse out cover glass with 0.01M PBS solution (pH7.4), wash chip 2 times with the 0.01M PBS solution (pH7.4) that contains 0.3%Tween 20, each 10 minutes.The centrifuge dripping chip detects with gene chip scanning instrument scanning, the record fluorescent signal.

The fluorescent signal that is write down is compared with the fluorescent signal of the preceding scanning of experiment, obtain the fluorescent signal difference, according to albumen gradient and fluorescent value production standard curve.

4, detect transcription factor NF-KB (nuclear extract) with the Label-dsDNA micro-array chip

5-10 μ g is contained the proteic nuclear extract of transcription factor NF-KB (HeLa Nuclear Extract, TNF-α-stimulated, 20min) be diluted in DNA binding buffer liquid [10mM HEPES pH7.9,50mM KCl, 2.5mM DTT, 0.1mM EDTA, 0.05%NP-40,10% Glycerol, 5%fetal bovine serum, 0.05mg/ml poly (dI-dC)] in, 37 ℃ hatch 10 minutes after, volume with every array 10 μ l is added on the Label-dsDNA micro-array chip, and 37 ℃ were continued to hatch 50 minutes.(pH7.4) carefully rinses out cover glass with 0.01MPBS solution, washs chip 2 times with the 0.01M PBS solution (pH7.4) that contains 0.3%Tween 20, each 10 minutes.

With exonuclease III (Fermentas Life Science) reaction solution (1U/ μ l Exonuclease III, 66mM Tris-HCl, 30 ℃ of pH8.0at, 0.66mM MgCl ₂) be added on the Label-dsDNA micro-array chip with the volume of every array 10 μ l, hatched 10 minutes for 37 ℃.Carefully rinse out cover glass with 0.01M PBS solution (pH7.4), wash chip 2 times with the 0.01M PBS solution (pH7.4) that contains 0.3%Tween 20, each 10 minutes.

The fluorescent signal that is write down is compared with the fluorescent signal of the preceding scanning of experiment, obtain the fluorescent signal difference.Carry out quantitatively according to the protein standard curve.

Claims

1, exonuclease III digestion Label-dsDNA micro-array chip detects transcription factor protein, has proposed a kind of detection transcription factor novel method, it is characterized in that using this method detection transcription factor protein expression and activation levels to comprise the steps:

A) prepare the Label-dsDNA micro-array chip;

B) transcription factor protein and Label-dsDNA micro-array chip reaction;

C) exonuclease III and Label-dsDNA micro-array chip reaction;

D) the Label-dsDNA micro-array chip is carried out check and analysis.

2, exonuclease III digestion Label-dsDNA micro-array chip according to claim 1 detects transcription factor protein, the Label-dsDNA micro-array chip that it is characterized in that preparation in the step (a) is made of chip and the Label-dsDNA probe molecule that is fixed on the chip;

3, Label-dsDNA probe molecule according to claim 2 is characterized in that it is the double stranded DNA molecule that forms after the single stranded nucleic acid molecule A of two base sequence reverse complementals and the B renaturation;

4, nucleic acid molecule A according to claim 3 is characterized in that its 3 ' end has carried out corresponding chemically modified according to the chemical group of chip surface, so that the Label-dsDNA probe molecule is affixed to chip surface by chemical reaction;

5, nucleic acid molecule A according to claim 3 and B, after it is characterized in that A and B annealing, terminal 5 ' the end with A of 3 of B ' can concordant, recessed or outstanding 1 to 4 Nucleotide, so that exonuclease I II is when acting on the Label-dsDNA probe molecule, and can be from the terminal B that degrades of 3 of B ';

6, nucleic acid molecule B according to claim 3 is characterized in that the terminal or close 5 ' end of 5 of B ' contains the Nucleotide that special marking is modified;

7, the Nucleotide of special marking modification according to claim 6 is characterized in that special marking is that fluorescein, digoxin, vitamin H etc. can rely on its chemical property to carry out the chemical molecular of check and analysis;

8, nucleic acid molecule A according to claim 3 and B is characterized in that containing the transcription factor protein binding sequence on the Label-dsDNA probe molecule of A and B annealing back formation;

9, transcription factor protein binding sequence according to claim 8, it is characterized in that it on the nucleic acid molecule B between Nucleotide and 3 ' end that special marking is modified, when transcription factor protein combines with the Label-dsDNA probe molecule, transcription factor protein can stop the digestion of exonuclease I II, and the Nucleotide that the protection special marking is modified is not digested;

10, chip according to claim 2, it is characterized in that it is the solid support that has rigidity and semi-rigid surface, and physical or chemical treatment has been carried out on the solid support surface, makes its surface have certain chemical group, so that being connected and fixed of Label-dsDNA probe molecule;

11, Label-dsDNA probe molecule according to claim 10 is connected and fixed, it is characterized in that between chemical group that chemical group that 3 of A in the Label-dsDNA probe molecule ' is end modified and chip surface modify chemical reaction taking place, the Label-dsDNA probe molecule can be fixed firmly to chip surface;

12, exonuclease III digestion Label-dsDNA micro-array chip according to claim 1 detects transcription factor protein, it is characterized in that in the step (b) transcription factor protein with the reaction of Label-dsDNA micro-array chip, be the transcription factor protein in various sources, comprise transcription factor protein, the separation and purification from the cell or tissue lysate of artificial expression preparation transcription factor protein, contain the cell or tissue extract of transcription factor protein;

13, exonuclease III digestion Label-dsDNA micro-array chip according to claim 1 detects transcription factor protein, it is characterized in that the reaction of middle transcription factor protein of step (b) and Label-dsDNA micro-array chip, is specific recognition and bonded process between the fixed Label-dsDNA probe molecule of transcription factor protein and Label-dsDNA micro-array chip surface;

14, exonuclease III digestion Label-dsDNA micro-array chip according to claim 1 detects transcription factor protein, it is characterized in that the reaction of step (c) amplifying nucleic acid excision enzyme III reaction solution and Label-dsDNA micro-array chip, be that the reaction soln that will contain exonuclease III covers the Label-dsDNA microarray, make exonuclease III produce the process of endonuclease reaction to fixed Label-dsDNA probe molecule on the Label-dsDNA micro-array chip;

15, special marking according to claim 7, it is characterized in that when special marking is fluorescein, the check and analysis of in the step (d) the Label-dsDNA micro-array chip being carried out can be carried out check and analysis to the fluorescent signal of Label-dsDNA micro-array chip by fluoroscopic examination instruments such as fluorescent microscope, gene chip scanning instruments;

16, special marking according to claim 7, it is characterized in that when special marking is chemical moleculars such as digoxin, vitamin H, the check and analysis of in the step (d) the Label-dsDNA micro-array chip being carried out, can be earlier with fluorescently-labeled DigiTAb, biotin antibody or streptavidin etc. and the reaction of Label-dsDNA micro-array chip, relend fluoroscopic examination instruments such as helping fluorescent microscope, gene chip scanning instrument the fluorescent signal of Label-dsDNA micro-array chip carried out check and analysis.