CN1746318A

CN1746318A - Detection of DNA binding protein with exonuclease protective DNA probe and hybrid DNA microarray chip

Info

Publication number: CN1746318A
Application number: CN 200410041930
Authority: CN
Inventors: 王进科
Original assignee: Nanjing Xinyihua Group Co Ltd
Current assignee: Nanjing Xinyihua Group Co Ltd
Priority date: 2004-09-10
Filing date: 2004-09-10
Publication date: 2006-03-15

Abstract

The invention is to detect the DNA conjugated protein using the hybrid DNA micro-array slug of exonuclease to protect DNA probe. The process is: (a) preparing the combined protein with DNA probe, the vector PCR, Tag-DNA; (b) combining the combined protein with DNA probe and the DNA conjugated protein; (c) treating with the exonuclease; (d) PCR amplifying and marking; detecting the hybrid product of PCR and the Tag-DNA. The method can detect many kinds of the DNA conjugated protein simultaneously but not modifying and marking the protein.

Description

It is conjugated protein that exonuclease protection dna probe hybrid dna micro-array chip detects DNA

One, technical field:

DNA conjugated protein (DNA-binding protein) be bioprotein group (proteomics) thus in a class can combine the protein that produces critical function with DNA, this proteinoid is repaired in (DNA reparation), the DNA restriction cell processes (cellular processes) such as (DNA restriction) at gene expression regulation (gene expression regulation), DNA reorganization (DNA recombination), DNA and is played a significant role.When some and DNA interact in this proteinoid, present the dna sequence dna function of specific connecting, as take place with DNA that all kinds of DNA of dna sequence dna specificity bonded are conjugated protein, various restriction enzymes etc.Because the vital role effect that this proteinoid is brought into play in cell processes, they have become the important object of functional genome (functional genomics) and protein group (proteomics) research.Regulate because this proteinoid participates in numerous expression of gene, therefore have confidential relation, become the important target spot (drug target) of diagnosis, treatment and drug research with a lot of diseases.The check and analysis of DNA binding protein expression and activation degree are the main means of its function of research.This patent has proposed the novel method of a kind of DNA of detection binding protein expression and activation levels, this method will be for being molecular biology (molecular biology), functional genomics, the conjugated protein correlative study of DNA in proteomics and biomedicine (Biomedicine) field provides a kind of new check and analysis technology, can promote the conjugated protein relevant scientific research of DNA in these fields, and a kind of disease-related DNA is provided in biomedical sector protein-bonded diagnostic techniques, and DNA conjugated protein be drug screening (drug screening) technology of target spot.

Two, background technology:

DNA is conjugated protein to be a class important protein matter in the bioprotein group, is the important object of functional genome and proteome research; All there is confidential relation in numerous disease with protein-bonded unconventionality expression of DNA and activation, becomes the important target spot of disease treatment and drug research.The check and analysis of DNA binding protein expression and activation degree are the main means of its function of research.Therefore, related detection analyzing DNA binding protein expression and activation degree Study on Technology are subjected to the attention of scientific circles always.

Up at present, scientists has been set up the check and analysis technology of multiple DNA binding protein expression and activation degree based on this level of DNA/ protein interaction.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 the conjugated protein binding sequence (consensus of DNA, binding sites) two strands (double-stranded) oligonucleotide (oligonucleotides), and use the labelled with radioisotope oligonucleotide, with labeled oligonucleotide with contain the protein-bonded cell or tissue extract of DNA mix 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 DNA binding protein expression and activation degree.This technology still is used for the conjugated protein check and analysis of DNA 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 (nylonmembrane), 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 DNA binding protein 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 conjugated protein check and analysis of DNA.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 DNA binding protein expression of gel shift experiment fully on the technological thought angle and activation detects and analytical technology is very important.Therefore we are engaged in the conjugated protein detection Research on New of DNA for many years always, and have set up multiple double-stranded DNA micro-array chip technology (Chinese patent ZL02112780.8,02137945.9,03152881.3,03132206.9).In the patented technology that we have invented, protein-bonded detection is directly to prepare double-stranded DNA on chip to DNA, be fixed on the chip on the double-stranded DNA the protein-bonded binding site of DNA is chimeric, reaction by protein solution to be checked and double-stranded DNA chip, make protein bound on chip DNA, carry out the protein-bonded detection of DNA.This method needs to use fluorescein-labelled protein in detection, or prepare proteinic antibody and carry out mark with fluorescein and could realize chip detection, this not only requires experiments such as labelled protein, preparation antibody, and mark may cause the influence of DNA combined function to albumen.This method is on detecting a kind of protein and chip during the interaction between numerous DNA, and is very effective; But when detecting multiple protein, face certain difficulty.Particularly importantly, the object that needs in practice to detect usually is the nuclear extract (nuclearextract) of tissue (tissue), cell (cell) etc., it is very difficult carrying out fluorescent mark to such complex mixture, particularly conjugated protein to those low-abundance DNA, be in nuclear extract mark they are unusual difficulties.If adopt antibody in addition, then need to prepare the antibody of all proteins of required detection, and with fluorescent mark they, it is conjugated protein just to be used for the multiple DNA of high throughput testing, this approach is obviously difficult more.Thus, we put forth effort to improve the conjugated protein detection technique of existing DNA, wish to set up some otherwise effective technique more, promote the industrialization and the application of correlation technique.Among the present invention; we analyze (Exonuclease Protection Assay) technology with the exonuclease protection; PCR (Polymerase Chain Reaction) technology and dna microarray chip (DNA microarray chip) technology combine effectively; set up " it is conjugated protein that exonuclease protection dna probe hybrid dna micro-array chip detects DNA " technology; be used under the situation of labelled protein not; it is conjugated protein that (simultaneous) detects multiple DNA synchronously, promptly realizes the protein-bonded high-throughput of DNA (high throughput) analyzing and testing.

Three, summary of the invention:

(1), goal of the invention

The purpose of this invention is to provide a kind of the needs carries out any mark and just can high-sensitivity high-flux detects the protein-bonded new technology of DNA DNA is conjugated protein, is convenient to rely on the biochip technology platform to realize the protein-bonded efficient analysis of DNA.For the correlative study of the DNA conjugated protein (DNA-binding protein) in molecular biology (molecular biology), functional genomics (functional genomics), proteomics (proteomics) and the biomedical fields such as (biomedicine) provides a kind of new experimental technique, promote the conjugated protein relevant scientific research of DNA in these fields, and at protein-bonded clinical detection of DNA and drug screening research.

(2), technical scheme

This patent has proposed the new technology of a kind of DNA of detection binding protein expression and activation levels; i.e. " it is conjugated protein that exonuclease protection dna probe hybrid dna micro-array chip detects DNA " uses this technology can realize check and analysis to the DNA binding protein expression (expression) and (activation) level of activation.The core of this technology is (Exonuclease Protection Assay) technology, PCR (the Polymerase Chain Reaction) technology of nucleic acid amplification and dna microarray chip (DNAmicroarray chip) technology of high throughput testing bioinformation to be analyzed in the exonuclease protection of the conjugated protein DNA binding site of identification of dna combine dexterously, reaches non-marked (label-free) high-throughput (High throughput) of DNA binding protein expression and activation levels is analyzed.

When using this technical Analysis DNA binding protein expression and activation levels, at first according to the nucleotide sequence of the protein-bonded DNA binding site of DNA that will detect, as common motif (consensus), and requirement to the PB-Probe structure is analyzed in utilization exonuclease protection in the present technique, design and synthesize and have " flanking sequence---the conjugated protein binding site of DNA---the PCR primer binding site---sequence label---PCR primer binding site---the conjugated protein binding site of DNA---flanking sequence " the protein bound dna probe (being called for short " PB-Probe ") of structure; When detecting to DNA is conjugated protein, at first moles such as a plurality of PB-Probe are mixed, become PB-Probe solution, again the conjugated protein solution of DNA to be detected and PB-Probe solution are mixed in the binding buffer liquid of favourable and DNA/ protein bound, under suitable temperature of reaction, carry out the association reaction appropriate time; Add exonuclease (exonuclease) reaction solution in DNA/ protein bound reaction system, enzyme is cut (digestion) appropriate time under optimal temperature, adds stop buffer again and thoroughly stops the exonuclease enzyme and cut activity; Endonuclease reaction carries out the DNA purifying to reaction system after stopping, and the DNA behind the purifying as template (template), is carried out pcr amplification reaction with the PCR primer.In the reaction of DNA/ protein bound, if exist the DNA of corresponding PB-Probe conjugated protein in the solution to be checked, then DNA is conjugated protein will sequence-specific association reaction (sequence-specific binding) take place with the PB-Probe in the solution, form " PB-Probe/DNA is conjugated protein " mixture (complex), in this mixture, DNA is conjugated protein to be combined on the DNA binding site at PB-Probe two ends, formed a kind of physical barrier (physicalhindrance), when exonuclease digestion, then bonded DNA is conjugated protein has stopped the circumscribed reaction of exonuclease along PB-Probe, make the exonuclease PB-Probe that can not degrade go up the PCR primer binding site (PCR primer-binding site) and the sequence label (Tag sequence) of DNA binding site inboard, therefore in the PCR reaction, just can be realized amplification by the PCR primer binding site of the PB-Probe of the conjugated protein protection of DNA and sequence label; And when the DNA that does not have corresponding PB-Probe in the solution to be checked is conjugated protein, the PB-Probe of exposed (naked) will be subjected to the attack of exonuclease, thoroughly degraded takes place to PB-Probe in exonuclease, therefore in the PCR reaction, because of no PCR primer binding site and sequence label, PCR just can not increase product.Therefore, the PCR product have that it's too late and how much represented in the protein soln to be checked DNA protein-bonded have that it's too late what.For ease of detecting pcr amplification product, in pcr amplification, can use fluorescently-labeled mononucleotide (Mononuleotide) or primer (primer), the PCR product is carried out mark, again with fluorescently-labeled pcr amplification product and Tag-DNA microarray hybridization, should the photoscan picture obtain the quantity information of various PB-Probe in the PCR product according to chip, thus high-throughput judge in the protein soln to be checked DNA protein-bonded have that it's too late what.Tag-DNA micro-array chip as used herein is a kind of common single stranded DNA (single-stranded DNA, ssDNA) micro-array chip (microarray chip), but the fixed dna probe is to design at the sequence label on the PB-Probe (Tagsequence) specially on the chip, so these chip DNA probes (CD-Probe) can detect the quantity information of various PB-Probe in the PCR product.

Use that this technology for detection DNA is conjugated protein to comprise following five steps:

1. prepare PB-Probe, CD-Probe and Tag-DNA micro-array chip;

2. PB-Probe combines with DNA is conjugated protein;

3. the exonuclease enzyme is cut;

4. pcr amplification and mark;

5. PCR product and dna microarray chip hybridization detect.

Preparation PB-Probe is the protein-bonded the first step of utilization this technology for detection DNA, also is a very important step.For realizing that present technique detects the protein-bonded function of DNA, the design of PB-Probe and preparation have following array structure: 1. PB-Probe is double-stranded DNA (double-stranded DNA, dsDNA) molecule; 2. the protein-bonded binding site of more than one same DNA is respectively contained at the two ends of PB-Probe; 3. there is the PCR primer binding site the conjugated protein binding site of the DNA of PB-Probe inboard; 4. there is specific label (Tag) sequence between the two PCR primer binding sites of PB-Probe.Therefore the PB-Probe that uses among the present invention has such structure, promptly " flanking sequence---the conjugated protein binding site of DNA---the PCR primer binding site---sequence label---PCR primer binding site---the conjugated protein binding site of DNA---flanking sequence ".

PB-Probe is linearity (linear) double chain DNA molecule that the single stranded nucleic acid molecule by two base sequence reverse complementals constitutes.The single stranded nucleic acid molecule that constitutes the two base sequence reverse complementals of PB-Probe can rely on two kinds of approach to obtain, and the one, synthetic two single stranded oligonucleotides of utilization solid state chemistry by nucleic acid annealing, form double chain DNA molecule; The 2nd, design and synthesize and have " flanking sequence---the conjugated protein binding site of DNA---primer sequence 1---primer sequence 2 " or the PCR primer of " flanking sequence---the conjugated protein binding site of DNA---primer sequence 1 ", rely on pcr amplification reaction from the known natural biology genomic dna of sequence, increase and obtain the PB-Probe of needs.Can utilize special primer sequence 2 amplification preparation PB-Probe during pcr amplification, and in the conjugated protein detection of DNA, use at the primer sequence 1 synthetic universal PC R primer PB-Probe that increases easily at different dna fragmentations.If possible, can use primer sequence 1 in the PB-Probe preparation and in detecting, be cloned in the genomic DNA fragment that has in the universal primer binding sequence carrier as utilization and prepare PB-Probe.

Realize the protein-bonded detection of DNA in order to use the exonuclease protection to analyze (Exonuclease Protection Assay) technology; the dna probe that the present invention analyzes in (DNA footprinting) traditional DNA footprint has carried out important transformation; promptly respectively set up a conjugated protein binding site of DNA at the two ends of dna probe; between the conjugated protein binding site of two DNA, set up PCR primer binding sequence and sequence label with information detection functions; whether combining of the conjugated protein binding site of two DNA of protein and PB-Probe can rely on sour excision enzyme protection subsequently to analyze; pcr amplification and chip hybridization are indicated.The protein-bonded binding site of DNA can be to identify that from the natural biology genome for example, the proteic DNA binding site of p53 is outside the binding sequence of finding such as common motif (consensus)

The proteic DNA binding site of SP1 is

The proteic DNA binding site of NF-κ is Deng; Also can be it to be had the nucleotide sequence of good sequence specificity in conjunction with affinity through the DNA of experiment in vivo and vitro screening is conjugated protein.Because it is conjugated protein that some exonuclease such as exonuclease III cross the less DNA of binding site sometimes, on probe design, can set up more than one protein binding site at each end of PB-Probe, or the higher protein binding site of avidity is used in design.In the PB-Probe design, also should be noted that at two ends of PB-Probe and set up some flanking sequences, so that DNA is conjugated protein and the good combination of PB-Probe.The conjugated protein flanking sequence that do not need of some DNA, also can with the conjugated protein binding site generation of DNA good binding, in this under situation, also can reduce the Nucleotide number of flanking sequence or flanking sequence not.Combine with PB-Probe and realize its detection by predetermined behavior in order to guarantee that DNA is conjugated protein, a PB-Probe goes up except that special the conjugated protein binding site of DNA that set up at the PB-Probe two ends, and other sequences comprise in flanking sequence, PCR primer binding site and the sequence label all must not contain the conjugated protein binding site of DNA.Conjugated protein in order to detect different DNA, different PB-Probe contains the protein-bonded binding site of different DNA, and it is conjugated protein to need to detect how many kinds of DNA in detection architecture, just need design and synthesize the PB-Probe of corresponding kind.The PB-Probe that then contains all kinds in the probe solution that detects is so that the multiple DNA of (simultaneously) detection is conjugated protein synchronously.But, owing in detection, multiple PB-Probe mixed as probe solution and protein soln and carries out association reaction, therefore the conjugated protein binding site sequence of DNA on PB-Probe is unique on this PB-Probe not only, also must be unique on all PB-Probe in probe solution, special, two kinds of protein-bonded binding sites of DNA must not appear on promptly a kind of PB-Probe.

For the ease of carrying out pcr amplification and detection, the present invention has designed the PCR primer binding site in the conjugated protein binding site of the DNA of PB-Probe inboard.On a PB-Probe, the sequence of PCR primer binding site must be unique, promptly has only the PCR primer binding site can supply the combination of PCR primer, other sequences comprise flanking sequence, protein binding site and sequence label all can not with the PCR primer annealing.On a PB-Probe, two PCR primer binding site sequences of sequence label both sides can identical (5 ' → 3 '), also can be different; If different, when pcr amplification, just need two different primers, and one couple of PCR primers; If identical, then only need a primer, just as RAPD.Owing in detection, will how mix as probe solution and protein soln and carry out association reaction towards PB-Probe, therefore the PCR primer binding site sequence on PB-Probe is unique on this PB-Probe not only, also must be unique, special on all PB-Probe in probe solution.As seen, among the present invention if during to the different PCR primer binding site of different PB-Probe design, the PB-Probe kind is many more in the probe solution, it is many more then to need to design on the synthetic PCR primer, then the complicacy of multiplex PCR is big more, particularly will take into account the specificity of PCR primer binding site sequence and consistent Tm value, then can increase PB-Probe and PCR design of primers and synthetic difficulty biglyyer, therefore this design obviously is not the preferential selection of the present invention.Comparatively speaking, on all PB-Probe, use a pair of or general PCR primer binding site, then greatly simplified PB-Probe and PCR design of primers and synthetic, therefore using universal PC R primer binding site and universal PC R primer is the scheme that override of the present invention is taked.

In order to use the dna microarray chip to realize the protein-bonded high throughput testing of DNA, set up specific label (Tag) sequence between the two PCR primer binding sites of PB-Probe of the present invention.Sequence label is the same with protein binding site, and it is unique not being only required in last its sequence of same PB-Probe, also must be unique, special on all PB-Probe in probe solution.Like this, the target protein that could represent this PB-Probe to detect to uniqueness of the sequence label on PB-Probe.The design of PB-Probe sequence label should also be noted that its based composition except that sequence-specific, so that at the dna probe of different PB-Probe sequence label design Tag-DNA micro-array chip, particularly require all CD-Probe that consistent Tm value will be arranged.If PB-Probe is amplification preparation from genomic dna, the sequence label of tackling each PB-Probe carries out detailed protein binding site examination, stops to contain the protein-bonded binding site of any known DNA on the sequence label of all PB-Probe.For known genome of sequence and dna clone, this work is not difficult.

It is conjugated protein that utilization the present invention detects DNA, also need prepare the PCR primer, so that cut back amplification PB-Probe at the exonuclease enzyme.The preparation of PCR primer of the present invention goes out to follow outside all requirements of general PCR design of primers, also should synthesize by the sequence signature of the last PCR primer binding site of PB-Probe.As the front PB-Probe is gone up the elaboration that the PCR primer binding site designs, the present invention avoids carrying out multiple PCR design of primers and synthetic as far as possible, also avoids carrying out multiplex PCR, should be preferentially to use universal PC R primer.

It is that the present invention realizes the conjugated protein detection final step of DNA that the Tag-DNA micro-array chip detects pcr amplification product, also is the important step that embodies high-throughput feature of the present invention.The design of Tag-DNA micro-array chip and preparation have following feature: 1. fixing a large amount of strand chip DNA probe (Single-stranded CD-Probe) molecules on the Tag-DNA micro-array chip; The nucleotide sequence of 2. a large amount of CD-Probe molecules has nothing in common with each other, and it is unique that different its nucleotides sequences of CD-Probe molecule is listed in all CD-Probe molecules; 3. the nucleotide sequence of each CD-Probe molecule design corresponding the sequence label of a kind of PB-Probe, promptly a kind of CD-Probe molecule can with wherein chain hybridization annealing of the sequence label of corresponding PB-Probe; 4. the sequence label of the corresponding different PB-Probe of different CD-Probe molecule on the chip; 5. during the PB-Probe molecular hybridization of Tag-DNA micro-array chip and pcr amplification, different CD-Probe molecules can reflect a kind of existence of sequence label in the PCR product of PB-Probe molecule of correspondence whether reach quantity what; 6. in order to improve the accuracy of chip detection, can design more than one CD-Probe at the sequence label of a PB-Probe, the amount of corresponding PB-Probe in the PCR product be made accurate differentiation by the codominance of many CD-Probe signal and the homogeneity of strength of signal; 7. the Tag-DNA micro-array chip should be provided with positive and negative control CD-Probe.

In the dna microarray chip detection, generally will carry out fluorescent mark to the DNA that detects, again with chip hybridization, hybridization back chip is through the fluorescent scanning analysis, just can obtain the hybridization signal of each probe, and then judge detect among the DNA quantity information corresponding to the target DNA molecule of various chip probes.In the present invention, pcr amplification product is carried out check and analysis, also need in pcr amplification, carry out mark amplified production for ease of utilization Tag-DNA micro-array chip.If use pcr amplification mark altogether, just can in the pcr amplification reaction system, add fluorescein-labeled mononucleotide, as Cy3-dUTP, Cy3-dCTP etc., make in its DNA product chain that enters pcr amplification; Or use fluorescein-labeled PCR primer, and carry out pcr amplification reaction, make fluorescence molecule on the DNA product chain belt of pcr amplification.Behind PCR product and the Tag-DNA microarray hybridization, chip just can directly carry out fluorescent signal with instruments such as gene chip scanning instrument, fluorescent microscopes and detect through suitably washing in such cases.Can also adopt other marking methods to realize mark in addition,,, also can use the PCR product of marks such as DIG, vitamin H etc., mark PCR product as DIG-dUTP, Biotin-dUTP etc. as using the Nucleotide of band DIG, vitamin H etc. among the PCR; In such cases behind PCR product and the Tag-DNA microarray hybridization, chip is through suitably washing, use fluorescently-labeled DIG-antibody, biotin antibody or streptavidin etc. and Tag-DNA microarray hybridization again, chip carries out fluorescent signal with instruments such as gene chip scanning instrument, fluorescent microscopes again and detects after washing.Except mark PCR product, can also adopting not, the other technologies of mark PCR product realize that the signal behind PCR product and the Tag-DNA microarray hybridization reflects, as increasing by one section oligonucleotide at 5 of PCR primer ' end, 4 a plurality of dATP of insertion or dGTP that other bases are alternate on it, behind PCR product and the Tag-DNA microarray hybridization, contain Cy3-dUTP or Cy3-dCTP at sheet archaeal dna polymerase extension, also can reflect hybridization signal; But this kind situation requires the CD-Probe of Tag-DNA micro-array chip to fix 5 ' end, and 3 ' free end.In addition if one section oligonucleotide of 5 ' terminal increase of PCR primer, can be behind PCR product and Tag-DNA microarray hybridization, also available one fluorescently-labeled oligonucleotide and PCR primer 5 ' end increases oligonucleotide hybridizes reflection PCR product and Tag-DNA microarray hybridization signal again.Other technologies in addition, as 3D-DNADendrimers, Rolling Circle Amplification Technology (RCA), technology such as Au-DNA are carried out the reflection of PCR product and Tag-DNA microarray hybridization signal.

It is conjugated protein to be used to the conjugated protein DNA for various sources of the DNA that detects in the present technique, comprises that the DNA DNA conjugated protein, separation and purification from the cell or tissue lysate of artificial expression preparation is conjugated protein and contains the protein-bonded cell or tissue extract of DNA.At first in binding buffer liquid, hatch appropriate time with PB-Probe is conjugated protein with DNA in the detection in suitable temperature of reaction (as 37C), as 20 minutes to half an hour, to be that DNA is conjugated protein with PB-Probe the sequence specific recognition takes place in solution and combines in its effect.The amount of the PB-Probe that adds in the combination anchor will suit.Can add in same reaction system in the detection and detect the protein-bonded PB-Probe of different DNA, multiple DNA is conjugated protein with high throughput testing.

Conjugated protein at DNA with after PB-Probe sequence-specific takes place combines, the exonuclease reaction solution is added in the reaction system, make exonuclease digestion reaction take place to PB-Probe, thoroughly degraded not with the conjugated protein bonded PB-Probe of DNA, make the PB-Probe of degraded in pcr amplification reaction subsequently, not increase.Digestion exonuclease that PB-Probe uses is that have on double-stranded DNA can be from 3 ' → 5 ' or 5 ' → 3 ' circumscribed dna digestion discharge the enzyme of mononucleotide one by one, as enzymes such as Ba131, exonuclease III, E.coli DNA Polymerase I, T4 phage DNA Polymerase, T7 phage DNAPolymerase, λ phage exonuclease.Selecting for use of enzyme, should select those reaction efficiency height as far as possible, reaction conditions is near the exonuclease of DNA/ protein bound damping fluid character.When selecting exonuclease for use, those enzymes that can carry out the circumscribed digestion of nucleic acid on strand and double-stranded DNA are more favourable.If select for use in the time of can only on double-stranded DNA, carrying out the enzyme of the circumscribed digestion of nucleic acid, as exonuclease III, need behind exonuclease reaction suitable time, to add the S1 nuclease and eliminate the single stranded DNA that exonuclease produces, carrying out pcr amplification.

Our experiment shows, exonuclease III is an operable unusual ideal exonuclease in the technology of the present invention, this enzyme has 3 of double-stranded DNA ' → 5 ' circumscribed activity, and all has stable activity in salt concn (0-100nM NaCl KCl alive) and pH (7.6-8.5) condition widely; This enzyme under (37 ℃) under general dna/protein bound temperature of reaction, have very high circumscribed activity (～500bp/ minute, Promega); In addition, general dna/protein bound damping fluid (has Mg ²⁺) just can become the feasible reaction conditions of this enzyme, therefore when carrying out the circumscribed reaction of exonuclease III, it is just passable only to add some enzymes, does not need to add other damping fluids again, so be fit to very much use in the exonuclease endonuclease reaction of the present invention.The digestive efficiency of exonuclease III is very high; at 37 ℃; per minute can excise 420 Nucleotide (Fermentas); therefore the time of enzyme reaction is generally very short; and that the life-span of DNA/ protein complex is generally reacted the needed time than exonuclease III is long; the adding of exonuclease III generally can not influence the DNA/ protein complex in addition, so exonuclease III is widely used in, and conventional DNA footprint is analyzed (DNAfootprinting) or the exonuclease protection is analyzed in (exonuclease IIIprotection assay).Another benefit of exonuclease III is, this enzyme is verified available conjugated protein with the DNA in the analysis of cells extract, this is highly beneficial to analyzing clinical sample, because clinical sample usually is tissue or cell, from these samples,, just can analyze with exonuclease III as long as extracting goes out nucleoprotein or cell protein.Therefore, exonuclease III is the exonuclease that override of the present invention uses.But when using this enzyme, can not use 3 ' end ratio, 5 ' distal process to go out the PB-Probe of 4 above Nucleotide, the PB-Probe of flush end and 3 ' recessed end is best.

Cause enzymic digestion to fall the PB-Probe that dynamically comes off after those protein bound owing in the exonuclease protection is analyzed, prolong the enzyme reaction meeting, cause occurring false negative result.Therefore add PB-Probe/ protein bound reaction system at exonuclease, behind the reaction appropriate time, should add termination reagent such as EDTA or heat and stop the exonuclease endonuclease reaction fully, and immediately reaction product is carried out the DNA purifying, eliminate protein such as the conjugated protein and exonuclease of DNA, so that pcr amplification.

Described to a kind of when treating that protein soln detects above in the present technique, to the PCR product with a kind of fluorescein-labelled and with the Tag-DNA microarray hybridization, the power by fluorescent signal reflects whether protein exists and reaches quantity.Except the detection of this mode, can also be to two parts of protein solns, as a kind of be normal tissue cell nucleoprotein extract, another kind is a morbid state histocyte nucleoprotein extract, compare detection, in such cases, a kind of albumen PCR product is with a kind of fluorescein-labelled (as Cy3), another kind of albumen PCR product is used another kind of fluorescein-labelled (as Cy5), at last two kinds of PCR products are mixed and Tag-DNA micro-array chip cohybridization, chip two channels fluorescence (Cy3, Cy5) after the scanning, stack Two Colour Fluorescence image is judged the rise (upregulate) or the downward modulation (downregulate) of protein expression or activation levels.

(3), technique effect

The conjugated protein class key protein that becomes present genome and the attention of protein groups institute because of the important cell functions such as regulation and control of being responsible for genetic expression of DNA, and the close relation that exists between numerous disease and conjugated protein unconventionality expression of DNA and activation, caused the concern of biomedicine field, the conjugated protein important target spot that has become disease treatment and drug research of DNA to the conjugated protein research of DNA.Therefore, it is very urgent to set up conjugated protein Function detection of high-throughput DNA and analytical technology.For this reason, the present invention proposes a kind of conjugated protein high throughput testing technology of DNA of non-protein labeling, it is conjugated protein to utilize present technique to detect DNA, can overcome some technological deficiencies of conjugated protein method of traditional detection DNA and double-stranded DNA micro-array chip, realizes the protein-bonded efficient analysis of DNA.

" it is conjugated protein that exonuclease protection dna probe hybridization Tag-DNA micro-array chip detects DNA " technology that the present invention proposes; exonuclease III protection analytical technology, pcr amplification technology and dna microarray chip technology are combined, set up the protein-bonded novel method of a kind of efficient detection analyzing DNA.Through our experimental study, the technology that the present invention proposes is workable, and experimental result repeatability is good.In applying, when we commercially produce with the situation that PB-Probe, CD-Probe and Tag-DNA micro-array chip and relevant reagent set are provided under, only just can finish protein-bonded Synchronization Analysis to many DNA through four experimental procedures.The conjugated protein detection new technology of DNA provided by the invention, can realize the protein-bonded high throughput testing of DNA by instruments such as conventional PCR instrument, gene chip sample applying instrument, gene chip scannings, these instruments are applied in a lot of laboratories, therefore are very easy to promoting the use of of present technique.The test kit that commercialization provides can be put into Application Areass such as scientific research, medical treatment, uses and the protein-bonded correlative study of DNA, diagnosis and drug screening.Therefore, this technology will provide a kind of new experimental technique for the protein-bonded correlative study of DNA in the fields such as molecular biology, functional genomics, proteomics, promote conjugated protein relevant scientific research of DNA and exploitation in these fields.

The DNA binding protein assay kit that the technology of the present invention is produced will protein-bonded drug screening research has very important using value at DNA in clinical assistant diagnosis and biomedicine.For example, the expression of the conjugated protein p53 of DNA, 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.

Four, description of drawings:

Fig. 1 exonuclease protection dna probe hybrid dna micro-array chip detects the conjugated protein techniqueflow of DNA

Explain: PB: protein bound (Protein Binding); ED: exonuclease digestion (Exonuclease Digestion); P: purifying (Purification); A: amplification (Amplification by PCR); CH: chip hybridization (Chip Hybridization); S: scanning (Scanning).

Fig. 2 pcr amplification experimental result

Fig. 3 Tag-DNA microarray hybridization experimental result

Five, embodiment

Be example only herein, the concrete of the technology of the present invention is described with the experiment for preparing the conjugated protein NF-κ of PB-Probe detection DNA B

Embodiment.

1, NF-κ B PB-Probe, PCR primer, Tag-DNA micro-array chip prepare NF-κ B PB-Probe:

Wherein _ _ be NF-κ B binding site,

Be PCR primer binding site,

Be the Tag sequence.

When NF-κ B PB-Probe prepares, with two oligonucleotide with 100 μ M concentration be dissolved in the TEN damping fluid (10mM Tris, 1mM EDTA, 0.1M NaCl, pH8.0) in; The mole such as oligonucleotide solution (molar) that will match mixes, and 95 ℃ of insulations 10 minutes slowly are cooled to 15～25 ℃.Is 5 μ M with TEN damping fluid dilution part PB-Probe stoste to concentration, and 4 ℃ of preservations are standby.

The PCR primer:

primerR：5′...GCTGGGCTGCTGC...3′ Tm：46.5 GC：76.9％

primerF：5′...CCGCTACGGCTCG...3′ Tm：47.5 GC：76.9％

The PCR primer tasteless nucleotide is dissolved in the water with 50 μ M concentration.

The Tag-DNA micro-array chip::

CD-Probe：5′...NH2-TTTTTTTTTTCTGCACTGCTCATTAATATACTTCTGG...3′positive

5′...NH2-TTTTTTTTTTCTGCATGTATAGAACATAAGGTGTCGC...3′negative?control

With the CD-Probe oligonucleotide with 100 μ M concentration be dissolved in carbonic acid buffer (0.1M carbonatebuffer, pH9.0).The CD-Probe oligonucleotide of the 50 μ M that carbonic acid buffer is diluted arrives aldehyde group modified slide (SuperAldehyde slide with PixSys5500 (Cartesian Technology Inc.) point sample again, Telechem) surface, point sample are placed in the wet box and use 300mM K ₂HPO ₄(pH9.0) on the wetted filter paper, the wet box of sealing 37 ℃ of incubations 30 minutes, was used 2 * SSC/1%SDS solution washing 2 minutes again, the simple drip washing of water, and N2 dries up.Chip is used sodium borohydride solution [0.25% (w/v) NaBH again ₄, 0.06M AcNa, pH5.2] and soaked slide 30 minutes, remove superfluous aldehyde radical, 1%SDS solution washing 2 minutes, the simple drip washing of water, N ₂Dry up.

2, NF-κ B PB-Probe and DNA conjugated protein NF-κ B's combines

With the conjugated protein NF-kB protein of 2 μ l (1gsu/ μ l) DNA [rhNF-κ B (p50), Promega)], 2 μ l, 5 * DNA binding buffer liquid (50mM HEPES pH7.9,250mM KCl, 12.5mM DTT, 0.5mM EDTA, 0.25%NP-40,50%Glycerol, 25%fetal bovine serum) and 2 μ l, 5 μ M NF-κ B PB-Probe mix, add 4 μ l water again, form DNA/ protein bound reaction solution.DNA/ protein bound reaction solution was hatched 30 minutes at 37 ℃.

3, exonuclease III reaction

With 2 μ l, 10 * reaction buffer [660mM Tris-HCl (30 ℃ of pH8.0 at), 6.6mM MgCl ₂] and 0.2 μ l 200U/ μ lExonuclease III (MBI Fermentas #EN0191) adds in the combination anchor, hatches 5 minutes for 37 ℃.In enzyme reaction solution, add 75 μ l S1 nuclease reaction solution [40.5mM potassium acetate (pH4.6), 0.34M NaCl, 1.35mMZnSO again ₄, 6.75%glycerol, S1 Nuclease 0.25U/ μ l), hatched 5 minutes for 37 ℃.Add 10 μ l S1 stop buffers (300mM Tris, 50mM EDTA), 10 minutes deactivation S1 Nuclease of 70 ℃ of heating.Use phenol: chloroform (1: 1), phenol: chloroform: each extracting terminated enzyme reaction solution of primary isoamyl alcohol (24: 1) once, again with ice ethanol sedimentation DNA[0.1 volume NaCl/glycogen solution (1.1M NaCl, 0.25mg/ml glycogen), 1 volume ethanol], last 1ml 75%-20 ℃ of ice washing with alcohol DNA once, the dry DNA precipitation is dissolved in DNA in the 10 μ l water.

4, pcr amplification reaction and mark

DNA with top purifying does template, and primerF and primerR are that primer carries out pcr amplification.100 μ l-PCR reaction systems are: 2 μ l template DNAs, 0.5 μ M primerR, 0.5 μ M primerF, 0.2mM dATP, 0.2mM dGTP, 0.2mMdTTP, 0.15mM dCTP, 0.05mM Cy3-dCTP (Amersham Pharmacia), 2mM MgSO ₄, 10mM KCl, 8mM (NH ₄) SO ₄, 10mM Tris-HCl, pH9.0,0.05%NP-40,0.05U/ μ l Taq (Shenergy Biocolor).The PCR program: 95 ℃ 2 minutes, 94 ℃ 30 seconds, 50 ℃ 45 seconds, 72 ℃ 1 minute the circulation 25 times, 72 ℃ 5 minutes.Use phenol: chloroform (1: 1), phenol: chloroform: each extracting PCR product of primary isoamyl alcohol (24: 1) is once iced ethanol sedimentation DNA[0.1 volume 3M KAc, 1 volume ethanol with-20 ℃ again], at last once with-20 ℃ of 75% ice washing with alcohol DNA, the dry DNA precipitation.DNA is dissolved in the 50 μ l water, and 95 ℃ were heated 10 minutes, and quenching is on ice preserved.

5, pcr amplification and marked product and Tag-DNA microarray hybridization detect

Get purified PCR product and 2 μ l, the 5 * standard hybridization solution [25 * SSC, 0.5%N-lauroylsarcosine, 0.1%SDS, 5%blocking reagent (Roche)] that is dissolved in the water of 2 μ l and mix, add water 6 μ l again.Hybridization solution is added drop-wise on the Tag-DNA micro-array chip, covers hybridization solution with cover glass.Chip is put into hybridizing box, and sealing was hatched 3 hours for 60 ℃.Take out chip, respectively wash chip once with 2 * SSC/0.1%SDS and 0.2 * SSC/0.1%SDS solution, the simple drip washing of water again, the 300rpm centrifugal chip is removed residual liquid on the chip.With gene chip scanning instrument (ScanArray ^Lite, PackardBioScience BioChip Technologies) obtain fluoroscopic image on, sweep parameter is: Cy3 passage, 90% laser intensity, 80%PMT gain, 5 μ m resolving power..The strength of signal of fluorescence pcolor picture is analyzed with microarray analysis software (QuantArray  microarray analysis software, Packard BioScience BioChip Technologies).

Claims

1, to detect DNA conjugated protein for exonuclease protection dna probe hybrid dna micro-array chip, proposed the protein-bonded novel method of a kind of detections DNA, it is characterized in that using this method detection DNA is conjugated protein comprising the steps:

A) preparation protein bound dna probe (being called for short PB-Probe), PCR primer and Tag-DNA micro-array chip;

B) PB-Probe combines with DNA is conjugated protein;

C) the exonuclease enzyme is cut;

D) pcr amplification and mark;

E) PCR product and Tag-DNA microarray hybridization detect.

2, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein; it is characterized in that the PB-Probe that step (a) is prepared, every kind of PB-Probe is the linear dsdna molecule that is made of nucleotide sequence assembly " the conjugated protein binding site of the flanking sequence → DNA → PCR primer binding site → sequence label → conjugated protein binding site → flanking sequence of PCR primer binding site → DNA ".

3, the conjugated protein binding site of the DNA of PB-Probe according to claim 2 is characterized in that a kind of PB-Probe only contains the protein-bonded binding site of a kind of DNA, and different PB-Probe contain the protein-bonded binding site of different DNA; In the dna sequence dna of all PB-Probe, the conjugated protein binding site of the DNA of a kind of PB-Probe only appears on the conjugated protein binding site of the DNA position of this kind PB-Probe, does not have the conjugated protein binding site of DNA of this kind PB-Probe in other PB-Probe dna sequence dnas.

4, the PCR primer binding site of PB-Probe according to claim 2, it is characterized in that a kind of PB-Probe can contain two PCR primer binding sites that sequence is similar and different, different PB-Probe also can contain the similar and different PCR primer binding site of sequence; In the dna sequence dna of all PB-Probe, the PCR primer binding site only appears on the PCR primer binding site position of PB-Probe, does not have the PCR primer binding site in other PB-Probe dna sequence dnas.

5, the sequence label of PB-Probe according to claim 2 is characterized in that different PB-Probe contain not homotactic sequence label; In the dna sequence dna of all PB-Probe, the sequence label of a kind of PB-Probe only appears on the sequence label position of this kind PB-Probe, does not have the sequence label of this kind PB-Probe in other PB-Probe dna sequence dnas.

6, the flanking sequence of PB-Probe according to claim 2 is characterized in that a kind of PB-Probe can contain two flanking sequences that sequence is similar and different, and different PB-Probe also can contain the similar and different flanking sequence of sequence; In the flanking sequence of all PB-Probe, there be not the conjugated protein binding site of DNA, PCR primer binding site and the sequence label of all PB-Probe.

7, to detect DNA conjugated protein for exonuclease according to claim 1 protection dna probe hybrid dna micro-array chip, it is characterized in that the PCR primer that step (a) is prepared, can with the PCR primer binding site specificity annealed combination of PB-Probe; According to the sequences Design of the last PCR primer binding site of PB-Probe, different PB-Probe can use different PCR primers, also can use identical PCR primer.

8, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein, the Tag-DNA micro-array chip that it is characterized in that preparation in the step (a), fixing a large amount of strand chip DNA probes (being called for short CD-Probe), the nucleotide sequence of each CD-Probe has nothing in common with each other, a kind of CD-Probe only anneals with the sequence label specific hybrid of a kind of PB-Probe, and different CD-Probe can anneal with different PB-Probe sequence label specific hybrids; The CD-Probe that detects all PB-Probe is arranged on the Tag-DNA micro-array chip.

9, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein; it is characterized in that PB-Probe combines with DNA is conjugated protein in the step (b); wherein PB-Probe is the equal amount of mixture of multiple PB-Probe, so as in one-time detection the protein-bonded information of the multiple DNA of synchronization gain.

10, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein; it is characterized in that PB-Probe combines with DNA is conjugated protein in the step (b); wherein DNA is conjugated protein is that the DNA in various sources is conjugated protein, comprises that the DNA DNA conjugated protein, separation and purification from the cell or tissue lysate of artificial expression preparation is conjugated protein and contains the protein-bonded cell or tissue extract of DNA.

11, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein; it is characterized in that PB-Probe combines with DNA is protein-bonded in the step (b), is generation sequence specific recognition and bonded process between the conjugated protein and PB-Probe of DNA.

12, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein; it is characterized in that exonuclease enzyme in the step (c) is cut is meant exonuclease is added in step (b) reaction product, makes exonuclease that the process of circumscribed digestion reaction take place from the two ends of PB-Probe.

13, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein; it is characterized in that the exonuclease in the step (c); for having the nuclease of exonucleolytic activity; these enzymes can be from 3 of double-stranded DNA ' or 5 ' circumscribed reaction takes place, discharge mononucleotide one by one.

14, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein, it is characterized in that step (d) pcr amplification and mark, be meant that with step (c) DNA product be template, the PCR primer for preparing with step (a) is the pcr amplification reaction that primer carries out; In the pcr amplification reaction system, can add the mononucleotide that marker is modified, or the PCR primer modified of marker, make mark on the DNA product band of pcr amplification.

15, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein; it is characterized in that step (e) PCR product and Tag-DNA microarray hybridization detect; be that the PCR product is mixed with hybridization solution; carry out hybridization with the Tag-DNA micro-array chip again, make PCR product and CD-Probe that sequence-specific annealed process take place.

16, exonuclease protection dna probe hybrid dna micro-array chip detection DNA according to claim 1 is conjugated protein; it is characterized in that step (e) PCR product and Tag-DNA microarray hybridization detect; behind PCR product and Tag-DNA microarray hybridization; to carry out the process that signal obtains according to the chemical property of PCR product mark.