CN1733934A - Exonuclease III digesting FRET-dsDNA microarray chip for detecting transcription factor protein - Google Patents
Exonuclease III digesting FRET-dsDNA microarray chip for detecting transcription factor protein Download PDFInfo
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Abstract
Disclosed is a transcription factor protein detection method through exonuclease III digestion FRET-dsDNA micro array chips, wherein the expression and activation levels of the transcription factor are analyzed through the following steps: (1) preparing FRET-dsDNA micro array chips, (2) reacting the transcription factor with FRET-dsDNA micro array chips, (2) reacting the exonuclease III with FRET-dsDNA micro array chips, (1) proceeding detection and analysis to the FRET-dsDNA micro array chips.
Description
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 dsDNA micro-array chip technology of many transcription factors high throughput testing is in demand.Thus; we put forth effort to improve this technology; with dna microarray chip technology, fluorescence resonance energy transmission (fluorescence resonance energy transfer; FRET) technology and exonuclease III protection analysis (ExoIII Protection Assay) technology combines, and has proposed the present invention's " exonuclease III digestion FRET-dsDNA micro-array chip detects transcription factor protein " technology.This technology adopts FRET technical mark dsDNA micro-array chip to prepare the FRET-dsDNA micro-array chip; again the high throughput testing function of FRET-dsDNA micro-array chip technology is in the same place 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 the FRET-dsDNA micro-array 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 dsDNA micro-array chip detection transcription factor protein, can solve the dsDNA micro-array chip The Application of Technology problem that we set up well.
Fluorescence resonance energy transmits (fluorescence resonance energy transfer, FRET) being also referred to as " F rster transmission ofenergy ", is the phenomenon that a kind of radiationless, quantum stage excitation energy of being carried out to acceptor (acceptor) by donor (donor) is transmitted.It is meant when a fluorescence molecule (being donor) when being subjected to exciting, the process that energy shifts to another fluorophor that closes on (being acceptor).But only have when overlapping less than the absorption spectrum of 10nm and acceptor and excitation spectrum when the distance between acceptor and donor, FRET could take place.And along with the increase of spectra overlapping, the efficient of FRET will increase, and the contingent distance of FRET also will increase simultaneously.The variation of fluorophor distance and spatial orientation can cause the change of FRET efficient.
FRET depends on the distance R between two fluorophores, and FRET is used for measuring erecting shop apart from the distance between protein, film and the macromole of 10～80 .FRET provides a kind of survey instrument of nanoscale, makes that the variable in distance with the microscopic examination nanometer level becomes possibility, for the interaction of people Real Time Observation biomacromolecule in live body provides an effective means.
Utilize FRET measure interactional intermolecular apart from the time, use following calculating.Be defined as by the energy transfer efficiency (E) of donor: E=R to acceptor
0 6/ (R
0 6+ R
6); Wherein: R is the distance between A, B, R
0It is a constant that comes by absorption and emmission spectrum calculating.E can (fluorescence intensity F) calculates, i.e. E=Fda/Fd, the fluorescence intensity when wherein the Fda=acceptor exists, the fluorescence intensity when the Fd=acceptor does not exist from fluorescence intensity.In case E is known, at R
0Under the situation about knowing, just can from first equation, calculate R.
FRET is verified to be one of the most powerful strong technology in all fluorescence techniques.FRET almost can be used for the analysis of form of ownership, and the user is monitored occur in the interaction on the macromole distance (1-10nm).Because transmission ofenergy depends on R
-6(R is the distance between donor and acceptor), life-span of donor (lifetime) and quantum yield (quantum yield) reduce, and acceptor fluorescence increases or activate (sensitized).In most of FRET used, donor was different with acceptor dye (dyes), in these cases, relied on the fluorescent emission of acceptor or the fluorescent quenching of donor to measure FRET.Must there be spectrographic overlapping (spectral overlap) in the dyestuff that uses among the FRET, as the FRET dyestuff of use to (pairs of dyes, donor-acceptor pairs) Cy3/Cy5, Cy5/Cy5.5, CFP (cyan fluorescent protein)/YFP (yellowfluorescent protein).Use the dyestuff of these types, can detect (examine) interaction of molecules widely (molecular interactions), as enzyme analysis (enzyme assays), immunoassay (immunoassays) and other binding events (binding events).
FRET also is that one of most important applications is molecular beacon field (molecular beacons) at first.Molecular beacon is exactly the oligonucleotide probe (oligonucleotide probes) that can detect specific nucleic acid in the solution.Dyestuff and quencher (quencher) closely cause dyestuff by the cancellation of transmission ofenergy institute near (close proximity).
FRET has been widely used in numerous research fields, comprising:
1. protein structure and structure picture (Structure and conformation of proteins);
2. protein complex spatial distribution and assembling (Spatial distribution and assembly ofprotein complexes);
3. receptor/ligand interaction (Receptor/ligand interactions);
4. immunoassay (Immunoassays);
5. unit molecule interaction (Probing interactions of single molecules);
6. the structure of nucleic acid and structure picture (Structure and conformation of nucleic acids);
7. PCR in real time analysis and SNP detect (Real-time PCR assays and SNP detection);
8. nucleic acid hybridization detects (Detection of nucleic acid hybridization);
9. primer extension analysis detects sudden change (Primer-extension assays for detecting mutations);
10. automated DNA order-checking (Automated DNA sequencing);
11. lipid profile and transhipment (Distribution and transport of lipids);
12. film convergence analysis (Membrane fusion assays);
13. membrane potential sensing (Membrane potential sensing);
14. give birth to fluorescin enzyme substrates (Fluorogenic protease substrates);
15. ring-AMP and zinc indicator (Indicators for cyclic AMP and zinc).
The application of FRET in the DNA/ protein interaction research also comes into one's own, this technology successfully has been used for researching DNA and protein interactions, wherein present topmost application is that research protein is when combining with DNA, the variation of dna structure, the DNA bending (bending) that causes as protein bound.Except this mainstream applications, the application of FRET in DNA combination/protein detection at present is mainly reflected on the following dual mode.The one, utilize protein that two DNA half sites drawing of donor and receptor marker is in the same place, produce the FRET effect, to detect proteinic whether the existence; The 2nd, utilize protein that the DNA of donor mark and the protein antibody drawing of receptor marker are in the same place, produce the FRET effect, to detect proteinic whether the existence and the DNA/ protein interaction.Method once major advantage be only to need two DNA half sites of donor and receptor marker can detect protein in detecting, experiment simply efficiently, specificity is good; But the difficulty that has the design of DNA half site, and donor and receptor marker are inserted in the protein binding site, may hinder proteinic normal combination in addition can not in conjunction with, and concerning the protein that those DNA binding sites are not grown, design dna half site difficulty more even may not, even may, such DNA half site is difficult to conjugated protein.Method two needn't the design dna half site, but need the preparation protein antibody, and need fluorescent-labeled antibody, and maximum difficulty is to design and to prepare by protein bound and produces the DNA of the antibody of donor (or acceptor) mark enough approaching on the space and acceptor (or donor) mark and be not easy, even very difficult.
Three, summary of the invention:
(1), goal of the invention
The purpose of this invention is to provide a kind of FRET-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 preparation, detection technique and the equipment of gene chip; 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 FRET-dsDNA micro-array chip of band FRET mark; 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 FRET-dsDNA micro-array chip detects transcription factor protein " uses this technology can realize high throughput testing analysis to the many transcription factor expression (expression) and (activation) level of activation.The core of this technology is with fluorescence resonance energy transmission (Fluorescence Resonance Energy Transfer; FRET) technology and the exonuclease III protection of identifying transcription factor protein DNA binding site are analyzed (Exonuclease III Protection Assay) technology and are applied to dexterously in the dsDNA micro-array chip technology that we study always; advantage by three kinds of technology combines, but has set up the new technology that a kind of high-throughput (high-throughput) detects transcription factor protein.
When using this technical Analysis transcription factor expression and activation levels, at first according to the nucleotide sequence of the DNA binding site of the transcription factor protein that will detect, as common motif (consensus), and FRET molecularity feature, design and synthesize dsDNA probe (being called " FRET-dsDNA probe "), prepare the FRET-dsDNA micro-array chip with the FRET-dsDNA probe again with FRET function.During detection, if have transcription factor protein in the solution to be checked, then transcription factor protein will with the FRET-dsDNA probe generation sequence-specific association reaction (sequence-specific binding) on the chip, form " FRET-dsDNA probe/transcription factor protein " mixture (complex).In this mixture, being combined in of transcription factor protein formed a kind of physical barrier on the FRET-dsDNA probe, when exonuclease III digests, then the bonded transcription factor protein has stopped the circumscribed reaction of exonuclease III along FRET-dsDNA probe free end, makes exonuclease III can not touch and the degrade Nucleotide of FRET mark; Therefore when fluoroscopic examination, there is FRET result, and when not having transcription factor protein in the solution to be checked, exposed FRET-dsDNA probe will be subjected to the attack of exonuclease III, thoroughly degraded takes place to the FRET-dsDNA probe in exonuclease III, discharge the mononucleotide of FRET mark, therefore when fluoroscopic examination, the FRET effect is lost.
Use this method detection transcription factor protein expression and activation levels to comprise the steps:
A) prepare the FRET-dsDNA micro-array chip;
B) transcription factor protein and FRET-dsDNA micro-array chip reaction;
C) exonuclease III and FRET-dsDNA micro-array chip reaction;
D) the FRET-dsDNA micro-array chip is carried out the fluoroscopic examination analysis.
The technological thought of above step comprises two parts, at first is to prepare the FRET-dsDNA micro-array chip, and next uses the FRET-dsDNA micro-array chip to detect transcription factor protein.
The preparation of FRET-dsDNA micro-array chip is the first step that realizes the technology of the present invention, also be a very important step, key issue comprise design and prepare the dsDNA probe of preparation FRET-dsDNA micro-array chip, transcription factor protein in conjunction with the reaction of FRET-dsDNA micro-array chip, exonuclease III to the good action of FRET-dsDNA micro-array chip etc.The dsDNA probe that is used for preparing the present invention's FRET-dsDNA micro-array chip structurally should satisfy following condition:
A) the dsDNA 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 dsDNA 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; Formed FRET-dsDNA probe stationary end (immobilizing end) by the side that 3 of the nucleic acid molecule A that contains the chemically modified group ' is terminal and 5 of nucleic acid molecule B ' is terminal;
E) after nucleic acid molecule A and the B annealing, 3 of B ' is terminal than terminal outstanding 1 to 4 Nucleotide of 5 of A ', or it is terminal concordant with 5 of A ', or 3 ' end of B is recessed, be called free end (free end), when exonuclease III acts on the FRET-dsDNA probe, can be from carrying out property of the 3 ' end degraded B of free end B;
F) form on the nucleic acid molecule A and B of FRET-dsDNA probe, A contains the Nucleotide of donor (donor) mark, and B contains the Nucleotide of corresponding acceptor (acceptor) mark; Wherein donor is the chemical group or the chemical molecular that can produce fluorescence; Acceptor can absorb the donor emitted fluorescence, both can be to absorb after the donor emitted fluorescence the no longer chemical group or the chemical molecular of emitting fluorescence, also can be to absorb after the donor emitted fluorescence chemical group or the chemical molecular of emitting fluorescence again; The Nucleotide number of donor and receptor marker is not limited in one, when the Nucleotide number of donor and receptor marker increases, helps improving the susceptibility of detection;
G) on the FRET-dsDNA probe, there is enough approaching space length in the Nucleotide of donor and receptor marker, guarantees can transmit (FRET) to acceptor generation fluorescence resonance energy when donor is stimulated;
H) on the FRET-dsDNA probe, the Nucleotide of donor and receptor marker is in inboardend, the transcription factor protein binding sequence is in free end, promptly forms the topology layout of " inboardend---Nucleotide of donor and receptor marker---transcription factor protein binding sequence---free end ".
The preparation of FRET-dsDNA micro-array chip among the present invention; its technological thought derives from us and the medium of utilization DNA bag quilt in double-stranded DNA micro-array chip Study on Technology and invention, the molecular biology is carried out the technology that utilization exonuclease III in the experimental technique, molecular biology of the conjugated protein affinity chromatography separation and purification of DNA carries out DNA protection analytical technology researching DNA/protein interaction, and the FRET technology that is used for interaction of molecules research.The key problem in technology of preparation FRET-dsDNA micro-array chip is how to design and synthesize the dsDNA probe with FRET function, and double chain acid molecule is 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.
The FRET-dsDNA probe is linearity (linear) double chain DNA molecule that is formed by single stranded nucleic acid molecule A and B annealing back.In order to make the FRET-dsDNA probe have the FRET function, when probe prepares, make single stranded nucleic acid molecule A contain fluorescently-labeled Nucleotide (Fluorescein labeled nucleotide), be called donor (donor), the oligonucleotide that contains dT-amino (dT-amino) as chemosynthesis, use fluorescein-labelled dT-amino again, then make single stranded nucleic acid molecule A contain fluorescently-labeled Nucleotide; The employed fluorescein of marker nucleic acid molecule A can be diversified fluorescein, as AMCA, FITC, FAM, TET, JOE, HEX, TAMRA, ROX, CY-3, CY-5 etc.
When probe prepares,, make single stranded nucleic acid molecule B contain the Nucleotide of corresponding black quenching group (quencher) or resonance fluorescence (Resonant Fluorophore) mark, be called acceptor (receptor) at the fluorescein of A institute mark.When using the Nucleotide of quenching group mark, the release wavelength (emissionwave) of the fluorescence that produces when the donor fluorescein of A mark is stimulated, the absorbing wavelength (absorbance wave) of overlapping quenching group with answering maximum area; As when the fluorescein FAM of A mark, B can mark quenching group DABCYL[4-(4[prime]-dimethylaminophenylazo) benzoic acid], when FAM excites with the 484nm wavelength and discharges the fluorescence of 516nm wavelength, because the absorbing wavelength of DABCYL is 453nm, just in time cover the release wavelength of FAM, this moment, fluorescent energy was absorbed by DABCYL, was converted into heat and discharged, and do not produce fluorescence, i.e. cancellation.The grey quenching group mark of single stranded nucleic acid molecule B, it also can be the oligonucleotide that first chemosynthesis contains dT-amino (dT-amino), use black quenching group mark dT-amino again, then make single stranded nucleic acid molecule B contain the Nucleotide of black quenching group mark: the employed black quenching group of marker nucleic acid molecule B can be varied, as DABCYL, BHQ etc. commonly used.
When and when using the fluorescence molecule mark single stranded nucleic acid molecule B that can resonate at the donor fluorescein of A institute mark, the excitation wavelength of the fluorescein molecule of mark B (acceptor) should farthest overlapping donor fluorescein molecule emission wavelength, and the maximum emission wavelength gap of donor and acceptor is the bigger the better.At this moment, when donor was stimulated, the fluorescence that is discharged just in time was subjected to bulk absorption, the acceptor so the generation fluorescence that is stimulated.Transcription factor protein is many more in the solution, protected and Nucleotide receptor marker that do not degraded by exonuclease III is many more, this moment is when exciting with donor maximum excitation wavelength, then detect high more signal, and detect low more signal at the maximum emission wavelength place of donor at the maximum release wavelength place of acceptor; On the contrary, transcription factor protein is few more in the solution, be subjected to the Nucleotide of receptor marker of exonuclease III degraded many more, this moment is when exciting with donor maximum excitation wavelength, it is low more then to detect signal at the maximum release wavelength place of acceptor, and high more at the detected signal in the maximum release wavelength place of donor.Therefore, when FRET-dsDNA probe during with two kinds of fluorescein molecule marks that the FRET effect can take place, can both strengthen (enhancement) and finish proteinic detection by the signal of measuring acceptor, also can finish proteinic detection, perhaps finish proteinic detection by the ratio (the ratio between two signals) between two kinds of signals of evaluation by the signal cancellation (quenching) of measuring donor.Rate signal (ratiometric signal) may be more useful, because it may reduce the fluorescent signal (trivial artifacts) of trickle artificial generation, such as the non-specific cancellation (nonspecific quenching) that in muddiness or absorbing medium (turbid or absorbing medium), produces.
When and when using the single stranded nucleic acid molecule B of resonance fluorescence mark at the fluorescein of A institute mark, the FRET D-A that the FRET-dsDNA probe is contained can be varied to (FRET donor-acceptor pairs), as FAM/TAMRA, TET/TAMRA, HEX/TAMRA, AMCA/fluorescien etc.The FRET D-A of labeling nucleic acid probe is that the excitation wavelength of acceptor fluorescence molecule should be farthest overlapping with the emission wavelength of donor fluorescence or non-fluorescence molecule to the foundation of selecting, and the maximum of donor fluorescence molecule and acceptor fluorescence molecule discharges the wavelength difference distance and is the bigger the better.Maximum emission wavelength as FAM is 521nm, and the maximum excitation wavelength of TAMRA is 560nm, and the emmission spectrum of FAM and TAMRA excitation spectrum exist overlapping; The maximum emission wavelength of TAMRA is 582nm in addition, and has bigger gap between the maximum emission wavelength 521nm of FAM, thus FAM-TAMRA can to constitute a pair of FRET D-A right.
Choose the FRET D-A to just preparing the first step of FRET-dsDNA probe, key is to want strict to design D-A to the locus in the FRET-dsDNA probe.Because FRET is the dependent interaction of a kind of distance, only when the distance between acceptor and donor during less than 10nm, FRET could take place.When preparation FRET-dsDNA probe, no matter selected D-A centering acceptor is that fluorescence molecule also is the quencher molecule of non-fluorescence, when the single-chain nucleic acid B annealing of carrying the single-chain nucleic acid A of donor and carrying acceptor forms double-stranded wire FRET nucleic acid probe, donor must be closely near acceptor, as use
Under the condition of the distance between acceptor and donor less than 10nm, when donor was stimulated, institute's emitted fluorescence just in time was subjected to bulk absorption, donor so be stimulated emitting fluorescence or cancellation fluorescence.
It is 1 that the right quantity of FRET D-A in the present technique on the FRET-dsDNA probe is not limited in quantity, and it is 1 that the quantity of transcription factor protein binding sequence also is not limited in quantity.Increase the right quantity of FRET D-A and can improve the susceptibility of detection, right as a plurality of FRET D-As are set in the nucleotide sequence between transcription factor protein binding sequence and inboardend, promptly form " inboardend---the FRET D-A is right---and the FRET D-A is right---the FRET D-A is right---transcription factor protein binding sequence---free end " the FRET-dsDNA probe of structure.A plurality of transcription factor protein binding sequences also can be set on the FRET-dsDNA probe, promptly form the FRET-dsDNA probe of similar " inboardend---the FRET D-A is right---transcription factor protein binding sequence---transcription factor protein binding sequence---free end " structure, the FRET-dsDNA probe of this structure, more favourable for the transcription factor protein that those binding sequences are lacked, can prevent reading over of exonuclease III.
In the present technique when on a FRET-dsDNA probe, be provided with a plurality of FRET D-As to the time, can be that a plurality of " D-As " are right, as
Deng, also can be that a donor is joined a plurality of acceptors, as long as the distance between each acceptor and donor satisfies the FRET requirement, as the situation of " acceptor---acceptor---donor---acceptor---acceptor ".
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 fixed FRET-dsDNA probe have with liquid phase in transcription factor protein combine active, the FRET-dsDNA probe that requirement is used for fixing has enough length, particularly on the FRET-dsDNA probe 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 FRET-dsDNA probe 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 fixed FRET-dsDNA probe 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 the FRET-dsDNA probe to be affixed to chip surface, make it can stand washing in the testing process and unlikely coming off, during preparation FRET-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, NOS groups) 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 FRET-dsDNA probe.
At the fixing FRET-dsDNA probe 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 fixed FRET-dsDNA probe; The 2nd, earlier with two base sequence complementary single stranded nucleic acid molecules renaturation in liquid phase, form the FRET-dsDNA probe, be added on the chip FRET-dsDNA probe fixedly connected again; 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 fixed FRET-dsDNA probe at last; In such cases, the Nucleotide of receptor marker can be arranged in the primer, and when adopting this mode to prepare the FRET-dsDNA micro-array chip, can rely on a universal primer and a single stranded DNA chip hybridization that contains receptor marker Nucleotide to extend again, reduce preparation cost significantly.First method is difficult to prepare the FRET-dsDNA micro-array chip that the present invention proposes, therefore preferably adopt back two kinds of technology to prepare the FRET-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 the FRET-dsDNA probe, 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 FRET-dsDNA probe 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 serumalbumin, BSA) and Tris solution deactivation N-oxygen succinimide ester group etc.
By above-mentioned techniqueflow, then prepared the FRET-dsDNA micro-array chip that can be used for the transcription factor protein detection.The quality of chip preparation has determined the FRET-dsDNA micro-array chip to detect the reliability of transcription factor protein.Therefore preparing high quality FRET-dsDNA micro-array chip is the key point that realizes transcription factor protein measuring ability of the present invention.
Prepared the FRET-dsDNA micro-array chip and provide instrument for the detection of transcription factor protein of the present invention.Utilize this FRET-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 (DNA binding buffer) of particular chemicals prescription, outside the FRET-dsDNA micro-array chip, hatch appropriate time, to hatch thing again is added on the FRET-dsDNA micro-array chip, under optimal temperature, continue to hatch appropriate time, transcription factor protein in the extract is combined with FRET-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 FRET-dsDNA micro-array chip, just can be added on the FRET-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 FRET-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 FRET-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 FRET-dsDNA micro-array chip of transcription factor protein, remove reaction solution, again with suitable washings washing FRET-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 FRET-dsDNA micro-array chip incubation reaction and thorough washing of transcription factor protein, exonuclease III reaction solution is added on the FRET-dsDNA micro-array chip hatches certain hour, make the DNA on exonuclease III and the FRET-dsDNA micro-array chip that endonuclease reaction take place; After endonuclease reaction finishes, remove endonuclease reaction liquid, again with suitable washings washing FRET-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 FRET-dsDNA micro-array chip has two kinds of situations, when the DNA on the FRET-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 5 ' end of nucleic acid molecule B of FRET-dsDNA probe stationary end or near receptor marker Nucleotide can not digestedly be released into reaction soln, promptly become the free mononucleotide; And when the DNA on the FRET-dsDNA micro-array chip does not form " DNA/ transcription factor protein " mixture with transcription factor protein, then the digestion of exonuclease III can arrive the Nucleotide of 5 of nucleic acid molecule B ' end or near receptor marker, the Nucleotide of receptor marker is then digested to be released in the reaction soln, becomes the free mononucleotide.Endonuclease reaction is removed exonuclease III reaction solution and is washed the FRET-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 FRET-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 FRET-dsDNA micro-array chip are hatched; " DNA/ transcription factor protein " mixture that forms is many more; the Nucleotide that is subjected to the receptor marker that transcription factor protein protection do not cut by exonuclease III enzyme on the corresponding FRET-dsDNA of the being retained in micro-array chip is then many more; the FRET effect is strong more during the chip fluoroscopic examination, makes between transcription factor protein and FRET signal to have the quantity dependence.
After endonuclease reaction finishes, behind chip process thorough washing and the centrifuge dripping, just can carry out fluorescent signal to chip and measure with instruments such as fluorescent microscope, laser confocal microscope, gene chip scanning instruments.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 FRET-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 FRET-dsDNA micro-array chip technology.Therefore; the technology that we adopt the present invention to propose is improved FRET-dsDNA micro-array chip technology; be about to dna microarray chip technology and exonuclease III protection analytical technology and combine, proposed " exonuclease III digestion FRET-dsDNA micro-array chip detects transcription factor protein " technology.This technology is incorporated into exonuclease III protection analytical technology in the FRET-dsDNA micro-array chip technology; avoided in the other technologies dependence, and reached simultaneously high throughput testing purpose well many transcription factor proteins to transcription factor protein antibody.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 FRET-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 FRET-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 FRET-dsDNA micro-array chip, go out output and can put into the FRET-dsDNA micro-array chip reagent kit that scientific research, medical or similar products application places can be used.Quality examination and analysis revealed, FRET-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 FRET-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 FRET-dsDNA micro-array chip synoptic diagram
Fig. 2 exonuclease III digestion FRET-dsDNA micro-array chip detects transcription factor experiment flow legend a
Explain: PBS: protein binding site (Protein Binding Site)
D: donor (Donor)
A: acceptor (Acceptor)
PB: protein bound (Protein Binding)
ED: exonuclease III degrade (ExonucleaseIII degrading)
W: washing (Washing)
S: scanning (Scanning)
Fig. 3 exonuclease III digestion FRET-dsDNA micro-array chip detects transcription factor experiment flow legend b
Explain: PBS: protein binding site (Protein Binding Site)
D: donor (Donor)
A: acceptor (Acceptor)
PB: protein bound (Protein Binding)
ED: exonuclease III degrade (ExonucleaseIII degrading)
W: washing (Washing)
S: scanning (Scanning)
Fig. 4 exonuclease III digestion FRET-dsDNA micro-array chip detects transcription factor experimental result exemplary plot
Explain: A: the fluorescent signal of the microarray that the fixing back of the nucleic acid molecule A point sample of band dT-FAM forms
B: the fluorescent signal among the nucleic acid molecule B of band dT-Dabcyl and the A after the microarray annealing
Among the C:B microarray with use the ExonucleaseIII digestion process after NF-κ B combines again after fluorescent signal
Five, embodiment
Only with the experiment of preparation FRET-dsDNA micro-array chip and detection transcription factor protein NF-κ B, example illustrates the embodiment of patent of the present invention herein.
1, the design of FRET-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 FRET-dsDNA micro-array chip of transcription factor NF-KB, must contain the common motif dna sequence dna of NF-κ B bonded on the FRET-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 FRET-dsDNA micro-array chip:
Wherein
Be dT-FAM,
Be dT-Dabcyl; The underscore sequence is a NF-κ B binding sequence.
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 FRET-dsDNA probe.With carbonic acid buffer (0.1M Na
2CO
3-NaHCO
3, pH9.0) part FRET-dsDNA probe dilution being become concentration is 20 μ M spotting solutions, 4 ℃ of preservations are standby.
2, FRET-dsDNA micro-array chip preparation
We select the aldehyde group modified slide preparation FRET-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 Schiff base 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 FRET-dsDNA micro-array chip that can be used for detecting transcription factor NF-KB.Bag should airtightly be kept at 4 ℃ of refrigerators by good chip.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 FRET-dsDNA micro-array chip
With transcription factor NF-KB albumen [rhNF-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 FRET-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 horse Lay acid buffer, 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 FRET-dsDNA micro-array chip with the volume of every array 10 μ l, cover solution with cover glass, hatched 10 minutes for 37 ℃.Carefully rinse out cover glass with horse Lay acid buffer, with containing 0.3%0.01M PBS solution (pH7.4) washing chip 2 times, 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 FRET-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 FRET-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 FRET-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 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.Carry out quantitatively according to the protein standard curve.
Claims (18)
1, exonuclease III digestion FRET-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 FRET-dsDNA micro-array chip;
B) transcription factor protein and FRET-dsDNA micro-array chip reaction;
C) exonuclease III and FRET-dsDNA micro-array chip reaction;
D) fluoroscopic examination of FRET-dsDNA micro-array chip is analyzed.
2, exonuclease III digestion FRET-dsDNA micro-array chip according to claim 1 detects transcription factor protein, the FRET-dsDNA micro-array chip that it is characterized in that preparation in the step (a) is made of chip and the FRET-dsDNA probe molecule that is fixed on the chip;
3, FRET-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 FRET-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 FRET-dsDNA probe molecule, and can be from the terminal B that degrades of 3 of B ';
6, nucleic acid molecule A according to claim 3 and B is characterized in that nucleic acid molecule A contains the Nucleotide of donor mark, and nucleic acid molecule B contains the Nucleotide of corresponding receptor marker;
7, donor according to claim 6 is characterized in that it is the chemical group or the chemical molecular that can produce fluorescence;
8, acceptor according to claim 6, it is characterized in that it can absorb the donor emitted fluorescence, it both can be to absorb after the donor emitted fluorescence the no longer chemical group or the chemical molecular of emitting fluorescence, also can be to absorb after the donor emitted fluorescence chemical group or the chemical molecular of emitting fluorescence again;
9, the Nucleotide of donor according to claim 6 and receptor marker, it is characterized in that on the FRET-dsDNA probe molecule, there is enough approaching space length in the Nucleotide of donor and receptor marker, guarantees can transmit (FRET) to acceptor generation fluorescence resonance energy when donor is stimulated;
10, nucleic acid molecule A according to claim 3 and B is characterized in that containing the transcription factor protein binding sequence on the dsDNA probe molecule of A and B annealing back formation;
11, transcription factor protein binding sequence according to claim 10, it is characterized in that it is on the nucleic acid molecule B between the Nucleotide and 3 ' end in receptor marker, the Nucleotide that is receptor marker is in 5 of nucleic acid molecule B ' end one side, and the transcription factor protein binding sequence is in 3 of nucleic acid molecule B ' end one side;
12, the Nucleotide of donor according to claim 6 and receptor marker, it is characterized in that on the FRET-dsDNA probe molecule, the Nucleotide of donor and receptor marker is between chip surface and transcription factor protein binding sequence, when transcription factor protein combines with the dsDNA probe molecule, transcription factor protein can stop the digestion of exonuclease I II, and the Nucleotide of protection donor and receptor marker is not digested;
13, 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 dsDNA probe molecule;
14, dsDNA probe molecule according to claim 13 is connected and fixed, it is characterized in that between chemical group that chemical group that 3 of A in the dsDNA probe molecule ' is end modified and chip surface modify chemical reaction taking place, the dsDNA probe molecule can be fixed firmly to chip surface;
15, exonuclease III digestion FRET-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 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;
16, exonuclease III digestion FRET-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 dsDNA micro-array chip, is specific recognition and bonded process between the fixed dsDNA probe molecule of transcription factor protein and dsDNA micro-array chip surface;
17, exonuclease III digestion FRET-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 dsDNA micro-array chip, be that the reaction soln that will contain exonuclease III covers the dsDNA microarray, make exonuclease III produce the process of endonuclease reaction to fixed dsDNA probe molecule on the dsDNA micro-array chip;
18, special marking exonuclease III digestion FRET-dsDNA micro-array chip according to claim 1 detects transcription factor protein, it is characterized in that the fluoroscopic examination of FRET-dsDNA micro-array chip is analyzed in the step (d), can carry out the fluorescent signal check and analysis by fluoroscopic examination instruments such as fluorescent microscope, gene chip scanning instruments.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101666805A (en) * | 2009-07-15 | 2010-03-10 | 苏州纳米技术与纳米仿生研究所 | Method for preparing specific protein detection chip |
| CN102262117A (en) * | 2011-04-27 | 2011-11-30 | 上海大学 | Bioelectrochemical sensor for detecting nuclear factor-kappa B and preparation method and application of bioelectrochemical sensor |
| CN106970229A (en) * | 2017-01-25 | 2017-07-21 | 南京医科大学 | A kind of transcription factor detection method amplified based on DNA silver nanoclusters molecular beacon and exonuclease III cycle signals |
-
2004
- 2004-08-13 CN CN 200410041684 patent/CN1733934A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101666805A (en) * | 2009-07-15 | 2010-03-10 | 苏州纳米技术与纳米仿生研究所 | Method for preparing specific protein detection chip |
| CN102262117A (en) * | 2011-04-27 | 2011-11-30 | 上海大学 | Bioelectrochemical sensor for detecting nuclear factor-kappa B and preparation method and application of bioelectrochemical sensor |
| CN106970229A (en) * | 2017-01-25 | 2017-07-21 | 南京医科大学 | A kind of transcription factor detection method amplified based on DNA silver nanoclusters molecular beacon and exonuclease III cycle signals |
| CN106970229B (en) * | 2017-01-25 | 2018-11-16 | 南京医科大学 | A kind of transcription factor detection method based on DNA- silver nanoclusters molecular beacon and the amplification of exonuclease III cycle signal |
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