HK1079552B - A biochip-based method for nucleic acid capable of combining specific sequence binding proteins assay - Google Patents

Description

Method for detecting nucleic acid binding protein capable of binding specific sequence based on biochip

Technical Field

The invention relates to a detection method of nucleic acid binding protein capable of binding with specific sequence, in particular to a method for detecting nucleic acid binding protein capable of binding with specific sequence based on biochip.

Background

Nucleic acid binding proteins include double-stranded DNA binding proteins, single-stranded DNA binding proteins, RNA binding proteins, and the like.

Double-stranded DNA binding proteins are protein molecules or protein molecule complexes that specifically bind to a double-stranded DNA having a specific sequence. Double-stranded DNA binding proteins include mainly repressors in prokaryotes, operator proteins, transcription factors in eukaryotes, and the like. These double-stranded DNA binding proteins activate, inhibit, attenuate, or enhance the expression of a particular gene by binding to a DNA double strand (operator/promoter) of a particular sequence. The functions of repressor protein and operator protein in prokaryotes are relatively simple, and enzymes required by the metabolic process of coding cells and the expression of coding antibiotic tolerance genes can be regulated and controlled, so that the physiological activity of the cells is adaptive to the external environment; eukaryotic transcription factors have a wide variety of functions, and various phenomena such as cell cycle, apoptosis and canceration are closely related to specific transcription factors. In the regulation network of gene expression in organisms, especially eukaryotes, the expression of genes encoding proteins is regulated at three levels, pre-transcriptional, post-transcriptional and post-translational, by transcription factor-mediated transcriptional activation, transcription, post-transcriptional modification (including cleavage, capping and tailing of RNA), translation, post-translational modification (including phosphorylation, glycosylation, acetylation, etc.), and the like.

Transcription factor-mediated transcriptional activation is very important as the first loop of the entire gene expression regulatory network. The stress response of organisms to the external environment is mostly achieved by turning on or off specific genes by specific transcription factors. Studies have shown that the expression of most eukaryotic genes is regulated by one or a few specific transcription factors. The more complex the organism, the more genes encoding transcription factors, and the more complex the mechanisms by which gene expression is regulated. It is predicted that more than 5% of the genes encoding proteins in the human genome encode transcription factors. Many transcription factors are closely related to cancer. For example, some transcription factors are products of protooncogenes that are expressed only in malignant tissues or enhance expression (e.g., FOS and C-Myc), and some transcription factors are expressed only weakly or not in malignant tissues (e.g., p53 and E2F). Therefore, if the expression level of some or all transcription factors of an organism at a certain time can be detected, and then the data of known transcription factor regulation genes are combined, the regulation information of the level before transcription can be obtained, and the regulation information can be possibly used for diagnosing whether tissues are cancerated, screening drug targets, researching mechanisms of cell stress response, observing activation or closing of signal channels of cells and the like.

The existing cDNA microarray technology can give data on the mRNA expression status of all transcription factor-encoding genes at the genome-wide level. However, only the active transcription factor can regulate the expression of the gene, the activity of the transcription factor is often regulated by various modifications such as phosphorylation, acetylation, glycosylation, etc., and the localization of the transcription factor in the cell also affects the activity of the transcription factor, so the amount of the active transcription factor is not directly related to the expression of the mRNA or protein level of the transcription factor. For example, the expression of the transcription factor Yin Yang 1(YY1) at mRNA and protein levels is nearly constant during various stages of the cell cycle, but the amount of active YY1 is regularly changing with the cell cycle. Therefore, cDNA microarrays are theoretically incapable of providing transcription factor expression profiling data of interest to biologists.

A conventional method for detecting "active" double-stranded DNA binding proteins (transcription factors) is gel retention (EMSA: Electrophoretic Mobility Shift Assay, gel Shift, band Shift). Mixing the protein sample to be detected with known DNA double-stranded molecules with isotope labels for reaction, and then carrying out polyacrylamide gel electrophoresis under a non-denaturing condition. Because the migration of the protein-bound double-stranded nucleic acid molecules is slower than that of the double-stranded nucleic acid molecules not bound to the protein in the electrophoresis process, separate electrophoresis bands can be obtained on the film through autoradiography after electrophoresis, and whether the expected interaction between the double-stranded nucleic acid and the binding protein occurs can be judged. In recent years, gel retention techniques have also been improved, for example, by using fluorescence or chemiluminescence techniques instead of isotope detection. The detection of DNA-binding protein complexes using antibodies to specific double-stranded DNA binding proteins is directed to the phenomenon of non-specific binding and is called Super-shift. The advent of gel-retention technology has greatly pushed the study of DNA and protein interactions, but it has significant drawbacks: the operation is complex; the time and the labor are consumed, and the experiment is usually carried out in the whole day; the detection flux is low, only one double-stranded nucleic acid binding protein can be detected at one time, and the required sample amount is large when multiple double-stranded nucleic acid binding proteins are detected; if the detection is carried out by using isotope, the detection has potential harm to human body, and if chemiluminescence or fluorescence is used, the detection is expensive.

BD Biosciences Clontech, USA, introduced Mercury (Palo Alto, Calif.)^TMTranscription factor kit (Mercury)^TMTransformation Factor Kit). The kit gives a 96-well plate for the detection of transcription factors. Each well of a 96-well plate is coated with a double-stranded DNA probe having a nucleic acid sequence that specifically binds to a transcription factor. And adding the sample to be detected into the hole for reaction, and combining the probe with the corresponding transcription factor in the sample to be detected. After the cleaning, the water is added with the water,and adding primary antibody for specifically recognizing the transcription factor, adding enzyme-labeled secondary antibody for combination with the primary antibody, and performing chemiluminescence detection. The technology greatly shortens the experimental time of the traditional gel retention method, and replaces isotopes which may cause harm to human bodies by using a chemiluminescence technology. However, there still remains the problem of low throughput, i.e.only one transcription factor can be detected at a time (reaction in one well); when detecting multiple double-stranded nucleic acid binding proteins, a large amount of samples are required; the reaction requires the use of antibodies that specifically recognize transcription factors, most of which do not have commercial primary antibodies that meet the needs of the above-described methods.

There are also sequence-specific single-stranded DNA-binding proteins and RNA-binding proteins in organisms, most of which regulate the physiological activities of the organism. In addition, antibody-like nucleic acid ligands that specifically bind to target protein molecules can be screened by in vitro evolution (in vitro evolution) methods, and these nucleic acid binding proteins have not been amenable to high throughput assays.

Disclosure of Invention

The invention aims to provide a method for detecting nucleic acid binding protein based on a biochip, which has high detection flux and high sensitivity and specificity.

The invention provides a method for detecting nucleic acid binding protein based on a biochip, which comprises the following steps: 1) adding a solution containing a plurality of groups of nucleic acid capture probes into a biological sample system containing a plurality of nucleic acid binding proteins to be detected to form a nucleic acid capture probe-nucleic acid binding protein complex, wherein the nucleic acid sequence of the nucleic acid capture probe contains at least one binding sequence capable of being combined with the target nucleic acid binding protein; 2) separating the nucleic acid capture probe-nucleic acid binding protein complex and recovering the nucleic acid capture probe; 3) hybridizing the nucleic acid capture probe recovered in the step 2) with a plurality of single-stranded immobilized probes which are immobilized on a biochip substrate and correspond to the nucleic acid capture probe, wherein the nucleic acid sequence of the immobilized probes is complementary with the nucleic acid capture probe or one strand of the nucleic acid capture probe corresponding to the immobilized probes; 4) and detecting the hybridization result.

Wherein, the separation of the nucleic acid capture probe-nucleic acid binding protein complex in the step 2) is carried out according to the following five processes: a) separating the mixed sample obtained in the step 1) by using conventional gel electrophoresis, and obtaining the nucleic acid capture probe-nucleic acid binding protein complex by cutting gel, separating by using a kit or performing electroelution. b) Separating the mixed sample obtained in the step 1) by adopting a chromatographic column method to obtain a nucleic acid capture probe-nucleic acid binding protein complex. c) And (2) separating the mixed sample obtained in the step 1) through a filter membrane to obtain a nucleic acid capture probe-nucleic acid binding protein complex, wherein the filter membrane can adsorb proteins. d) Adding an antibody capable of specifically recognizing each nucleic acid binding protein into the mixed sample obtained in the step 1), and separating the nucleic acid capture probe-nucleic acid binding protein complex by using a method similar to antibody purification (for example, by using protein A/G coated agarose bead-bound antibody). e) Separating the mixed sample obtained in the step 1) by using a capillary electrophoresis device, and automatically collecting the nucleic acid capture probe-nucleic acid binding protein complex.

The method of the present invention may be named as "end labeling method", and may be used in detecting nucleic acid binding protein, such as double stranded DNA binding protein, single stranded DNA binding protein, RNA binding protein, etc. and non-natural nucleic acid ligand capable of binding protein molecule, such as thrombin (aptamer), etc. may be also detected through in vitro evolution process.

In the present invention, the nucleic acid-binding protein is preferably a double-stranded DNA-binding protein, and more preferably a transcription factor such as AP1, Sp1, p53, E2F, and the like.

When the method of the invention is applied to detection, the preferable conditions are as follows: the nucleic acid sequence of the nucleic acid capture probe comprises a binding sequence capable of binding to the target nucleic acid binding protein; one nucleic acid strand of each set of said nucleic acid capture probes has an overhang sequence at one end.

In order to increase the hybridization capacity of a nucleic acid capture probe to an immobilized probe, the immobilized probe is completely complementary to its corresponding nucleic acid strand with an overhang sequence in the nucleic acid capture probe.

In order to facilitate the detection of hybridization signals, the nucleic acid strand with the overhang sequence in the nucleic acid capture probe is further provided with a labeling molecule at one end, wherein biotin, digoxigenin, a fluorescent label, a quantum dot, a gold particle and a nanoparticle are preferred.

In order to increase the detection sensitivity, the method of the present invention may be modified as follows ("single-stranded amplification method before hybridization"): and (3) amplifying said recovered nucleic acid capture probe with a primer that hybridizes to a nucleic acid strand of said nucleic acid capture probe having a 3 'overhang sequence prior to said hybridizing of step 3), such that said primer hybridizes to a nucleic acid strand of said nucleic acid capture probe having a 3' overhang sequence to facilitate a subsequent nucleic acid amplification step.

Among them, the preferable cases are: the 3 ' overhang sequence is the same in one nucleic acid strand bearing a 3 ' overhang sequence of each set of said nucleic acid capture probes, and the sequence of said primer is fully complementary to said 3 ' overhang sequence.

In this case, for the convenience of detecting hybridization signals, a labeling molecule may be added to the system, the end of the primer molecule may be provided with the labeling molecule before amplification, or the amplification raw material may be doped with nucleotide with the labeling molecule during amplification, wherein the labeling molecule is preferably biotin, digoxigenin, a fluorescent label, a quantum dot, a gold particle, or a nanoparticle.

To increase the detection sensitivity, the method of the present invention may be further modified ("double-stranded amplification method before hybridization"): each set of said nucleic acid capture probes further comprising a 3 'overhang sequence and a 5' overhang sequence at the 3 'end and the 5' end of one nucleic acid strand, respectively; prior to said hybridizing of step 3), amplifying said recovered nucleic acid capture probe with two primers, one of said two primers being capable of hybridizing to the 3 'overhang of the nucleic acid strand of said nucleic acid capture probe having a 3' overhang sequence and a5 'overhang sequence, and the other primer having a sequence identical to the 5' overhang of the nucleic acid strand of said nucleic acid capture probe having a 3 'overhang sequence and a 5' overhang sequence.

Among them, the preferable cases are: the 3 'overhang sequence in the nucleic acid strand with a 3' overhang sequence and a5 'overhang sequence of the nucleic acid capture probe is the same, the 5' overhang sequence in the nucleic acid strand with a 3 'overhang sequence and a 5' overhang sequence of the nucleic acid capture probe is the same, one of the two primers is capable of hybridizing to the 3 'overhang of the nucleic acid strand with a 3' overhang sequence and a5 'overhang sequence in the nucleic acid capture probe, and the sequence of the other primer is identical to the 5' overhang of the nucleic acid strand with a 3 'overhang sequence and a 5' overhang sequence in the nucleic acid capture probe.

The operation flow of detection by double-strand amplification is shown in FIG. 7, wherein a capture probe 1 and a target protein 3 (circular and triangular in the figure) are mixed for reaction, a nucleic acid capture probe-nucleic acid binding protein complex 4 is obtained after separation, and the capture probe is recovered; and (3) amplifying the recovered capture probe by using two primers 6, hybridizing the amplified product with a chip 5 on which the immobilized probe 2 is immobilized, and detecting a hybridization signal to obtain a detection result. The preferred structures of the capture probe, immobilized probe and primer used are shown in FIG. 8: the capture probe contains a binding sequence for the protein of interest, one of its two strands having a 3 'overhang and a 5' overhang; the sequence of primer 1 is complementary to the 3 'overhang sequence and the sequence of primer 2 is identical to the 5' overhang; the immobilized probe sequence is identical to the binding sequence.

Similarly, for convenient hybridization signal detection, a labeling molecule can be added into the system, the end of the primer molecule can be provided with the labeling molecule before amplification, or the amplification raw material can be doped with nucleotide with the labeling molecule during amplification, wherein the labeling molecule is preferably biotin, digoxigenin, fluorescent label, quantum dot, gold particle or nanoparticle.

The invention adopts the biochip method to detect nucleic acid binding protein, especially transcription factor, and the hybridization signal detection of the capture probe and the immobilized probe has the advantages of high detection sensitivity and high detection flux; moreover, when the two improved methods (single-strand amplification before hybridization and double-strand amplification before hybridization) are adopted, the primers can be labeled in advance, and molecular labeling modification of each group of nucleic acid capture probes is not needed, so that the experiment cost can be greatly reduced. The method can be widely applied to the fields of disease diagnosis, drug target screening, disease mechanism and the like.

Drawings

FIG. 1 is a schematic diagram of the operation of the present invention for detecting various nucleic acid binding proteins using the "end-labeling method";

FIG. 2 is a schematic diagram of the structure of the probe for detecting various nucleic acid binding proteins according to the present invention using the "end-labeling method";

FIG. 3A is a schematic diagram showing a dot matrix design of a chip according to example 1;

FIG. 3B is the results of the experiment in example 1 for simultaneous detection of three binding proteins;

FIG. 3C shows the results of the experiment for simultaneously detecting three binding proteins in example 1, wherein a competitive binding probe of AP1 was added to the binding reaction system;

FIG. 3D is the results of the experiment in example 1 for simultaneous detection of three binding proteins, with the addition of a competitive binding probe for NFkB to the binding reaction system;

FIG. 3E shows the results of the experiment for detecting three binding proteins simultaneously in example 1, wherein a competitive binding probe Sp1 was added to the binding reaction system;

FIG. 4 is a schematic diagram of the operation of the present invention for detecting a plurality of nucleic acid binding proteins using the "single-strand amplification method";

FIG. 5 is a schematic diagram of the structure of probes and primers for detecting various nucleic acid binding proteins by using the "single-strand amplification method" according to the present invention;

FIG. 6A shows the results of example 2 using the "single-strand amplification method" to simultaneously detect three binding proteins (AP1, NFkB, Sp 1);

FIG. 6B is the result of the same "end-labeling" assay as in example 1 in example 2;

FIG. 7 is a schematic flow chart of the operation of the present invention for detecting a plurality of nucleic acid binding proteins using a "double-stranded amplification method";

FIG. 8 is a schematic diagram of the structure of probes and primers for detecting various nucleic acid binding proteins by the "double-strand amplification method" according to the present invention.

Detailed Description

Example 1 Simultaneous detection of 3 nucleic acid-binding proteins AP1, NFkB and Sp1 Using a DNA chip (end-labeling method)

The operation flow of detection by using a terminal labeling method is shown in figure 1, a capture probe 1 and a target protein 3 (circular and triangular in the figure) are mixed and react, a nucleic acid capture probe-nucleic acid binding protein complex 4 is obtained after separation, the capture probe is recovered and then hybridized with a chip 5 fixed with an immobilized probe 2, and a detection result can be obtained by detecting a hybridization signal.

Wherein, the preferable probe structures of the capture probe and the immobilized probe are shown in FIG. 2: the capture probe contains a binding sequence for a protein of interest, one of the two strands of which carries an overhang and a labeling molecule, illustrated as biotin (biotin); the immobilized probe is complementary to the long strand in the capture probe.

Test materials:

the transcription factors AP-1(c-Jun) (# E3061), NFkB (p50) (# E3770) and Sp1(# E6391) were from Promega corporation (Madison, Wis.). The composition of the universal binding buffer was: 10mM Tris-HCl (pH 7.5), 4% glycerol, 1mM MgCl2, 0.5mM EDTA, 5mM DTT, 50mM NaCl, 0.05mg/mL poly (dI-dC) · (dI-dC). The electrophoresis buffer used was 0.5 XTBE (0.9M Tris base, 0.9M boric acid, 0.02M EDTA, pH 8.0). PBST (phosphate buffer, 0.1% Tween 20) was used as a wash. Agarose for electrophoresis was obtained from Biowest (Miami, FL). Bovine Serum Albumin (BSA) was purchased from Amresco (Solon, OH.) Cy 3-labeled streptavidin was purchased from Amersham Biosciences (Uppsala, Sweden).

The experimental steps are as follows:

preparation of DNA probes: all the probes were synthesized by Shanghai Boya, and the sequences of the probes are shown in Table 1 (in the table, AP-1-IP is an immobilized probe of AP1, AP-1-LP, AP-1-FP are capture probes of AP1, AP-1-CP and AP-1-FP are competitive binding probes of AP1, and other probes are similarly shown; the overhang sequence of the LP probe in this example is 5 '-CGGGA-3'). The immobilized probes in each group were first dissolved in water and placed in a 50% Dimethylsulfoxide (DMSO) solution to a final concentration of 10 micromoles per liter. The protein capture probes were all solubilized with water and the corresponding probes (FP and LP probes in each set of probes) were annealed to form duplexes at a final concentration of 60 nanomoles per liter.

B, preparation of DNA chip: the immobilized probes described above were spotted onto amino-modified slides using a PixSys5500(Cartesian Technologies, Irvine, CA) robot in the lattice pattern of fig. 3A with a spot spacing of 350 microns. After the sample application, the slide is baked for 1 hour in a temperature box at 80 ℃, and then the nucleic acid molecules fixed on the surface of the slide are glued by using an ultraviolet glue connection instrument under the energy of 250 mJ. In FIG. 3A, A1-A5 is an AP1 immobilized probe, B1-B5 is an NFkB immobilized probe, C1-C5 is an Sp1 immobilized probe, D1-D5 is a TFIID immobilized probe, E1-E5 is an NC immobilized probe, F1-F5 is an HC immobilized probe, A6, B6, C6, D6, E6 and F6 are spotting controls which are nucleic acid molecules with fluorescein, and after the spotting is finished, the glass slide is scanned, the spotting controls can be detected, and the step of spotting the glass slide is proved to be correct.

C. Configuration of nucleic acid-protein binding system: the three transcription factors (1.0. mu.g AP1, 300ng NFkB and 1.0. mu. gSp1) and the protein capture probes (i.e., the annealing products of LP and FP) were mixed and added to the universal binding buffer to bring the final concentration of the buffer to 1X. The reaction was combined at room temperature for 30 minutes. In the experiments described in FIGS. 3C, 3D, and 3E, competitive binding probes (CP) were also added to the reaction system.

D. Isolation of nucleic acid-protein binding System: a2% agarose gel and TBE buffer for electrophoresis were prepared in advance, and the mixture was precooled to 4 ℃. The above-mentioned binding reaction system was added to the well of the gel and electrophoresed at 120 volts for 20 minutes. The desired gel sections were cut out according to the position of the bromophenol blue indicator in the running buffer.

E. Recovery of nucleic acids: the nucleic acid in the gel obtained in the previous step was recovered using QIAGEN type II gel recovery kit from Qiagen, according to the product instructions.

F. Hybridization analysis of the chip: the nucleic acid obtained in the above step was applied to a DNA chip in a hybridization solution (HC-LP probe was added thereto) prepared in a 15. mu.l system containing 3XSSC and 0.1% SDS, and hybridized at 65 ℃ for 1 hour. The slides were then washed with 0.2XSSC and 0.1% SDS wash for 10 minutes at room temperature and spun dry at 1000 rpm. Blocking was performed by adding 1% BSA to the chip under 37 ℃ for 30 minutes. The slides were washed with PBST, room temperature for 10 minutes, and spun dry at 1000 rpm. 15 μ l of Cy 3-labeled streptavidin was added to the chip surface at a concentration of 1. mu.g/ml, and the reaction was carried out at 37 ℃ for 1 hour. The slides were washed with PBST, room temperature for 10 minutes, and spun dry at 1000 rpm. The image was scanned using a Scanarray 4000 scanner and the images were analyzed using GenePix. Wherein FIG. 3A is a dot matrix layout of a chip; FIG. 3B is the results of an experiment using the "end-labeling method" to simultaneously detect 3 nucleic acid binding proteins on a chip; FIG. 3C shows the results of an experiment using the "end-labeling method" to simultaneously detect 3 nucleic acid binding proteins on a chip, wherein a competitive binding probe to the nucleic acid binding protein AP1 was added to the binding reaction system; FIG. 3D is the experimental result of simultaneous detection of 3 nucleic acid binding proteins on a chip using the "end-labeling method", incorporating competitive binding probes for the nucleic acid binding protein NFkB into the binding reaction system; FIG. 3E shows the results of an experiment using the "end-labeling method" to simultaneously detect 3 nucleic acid-binding proteins on a chip, wherein competitive binding probes of nucleic acid-binding protein Sp1 were added to the binding reaction system. The result shows that the 'end labeling method' of the invention can obviously detect three transcription factors, and the sequence in NC is from the promoter sequence of the prokaryote phage and the binding sequence of the transcription factor of eukaryote is greatly different and can not be combined with any transcription factor, so the signal of NC probe is negative all the time; HC was used as a hybridization control and was added prior to hybridization to normalize the different arrays, so HC signals were always positive.

TABLE 1 Probe sequence Listing of immobilized probes and Capture probes for several transcription factors

Group number	Transcription factor	Probe numbering	Name of probe	Probe sequence
					1	AP-1	1	AP-1-IP	5′-T20-CGCTTGA TGAGTCAGCCGGA-TCCCG-3′
2	AP-1-LP	5′-biotin-CGGGA-TCCGGC TGACTCATCAAGCG-3′
			3	AP-1-FP			5′-CGCTTGA TGAGTCAGCCGGA-3′

		4	AP-1-CP	5′-TCCGGC TGACTCATCAAGCG-3′
					2	NFκB	5	NFκB-IP	5′-T20-AGTTGAG GGGACTTTCCCAGGA-TCCCG-3′
6	NFκB-LP	5′-biotin-CGGGA-TCCTG GGAAAGTCCCCTCAACT-3′
			7	NFκB-FP			5′-AGTTGAG GGGACTTTCCCAGGA-3′
8	NFκB-CP	5′-TCCTG GGAAAGTCCCCTCAACT-3′
			3	SP1			9	SP1-IP	5′-T20-AAAGC CCCGCCCCGATATAAT-TCCCG-3′
10	SP1-LP	5′-biotin-CGGGA-ATTATATC GGGGCGGGGCTTT-3′
					11	SP1-FP	5′-AAAGC CCCGCCCCGATATAAT-3′
12	SP1-CP	5′-ATTATATC GGGGCGGGGCTTT-3′
					4	TFIID	13	TFIID-IP	5′-T20-CGCCTACCTCATT TTATATGCTCTGC-TCCCG-3′
14	TFIID-LP	5′-biotin-CGGGA-GCAGAGCA TATAAAATGAGGTAGGCG-3′
			15	TFIID-FP			5′-CGCCTACCTCATT TTATATGCTCTGC-3′
16	TFIID-CP	5′-GCAGAGCA TATAAAATGAGGTAGGCG-3′
			5	NC			17	NC-IP	5′-T20-CTATGTGGTGAACTCCTCCTAAATA-TCCCG-3′
18	NC-LP	5′-biotin-CGGGA-CGGGATATTTAGGAGGAGTTTCACCACATAG-3′
					19	NC-FP	5′-CTATGTGGTGAACTCCTCCTAAATA-3′
6	HC	20						HC-IP	5′-T20-AGACGGAAGACATATGGCCGCTC-TCCCG-3′
			21	HC-LP	5′-biotin-CGGGA-GAGCGGCCATATGTCTTCCGTCT-3′

Example 2 Simultaneous detection of 3 nucleic acid-binding proteins AP1, NFkB and Sp1 Using a DNA chip (Single Strand amplification method)

The operation flow of detection by single-strand amplification is shown in figure 4, the capture probe 1 and the target protein 3 (circular and triangular in the figure) are mixed and reacted, a nucleic acid capture probe-nucleic acid binding protein compound 4 is obtained after separation, and the capture probe is recovered; and (3) amplifying the recovered capture probe by using a primer 6, hybridizing the amplified product with a chip 5 on which the immobilized probe 2 is immobilized, and detecting a hybridization signal to obtain a detection result.

The preferred design principle of the capture probe, immobilized probe and primer is shown in FIG. 5: the capture probe contains a binding sequence for the protein of interest, one of its two strands having a 3' overhang; primer sequences complementary to the 3' overhangs; the immobilized probe is identical to the binding sequence.

Test materials:

the transcription factors AP-1(c-Jun) (# E3061), NFkB (p50) (# E3770) and Sp1(# E6391) were from Promega corporation (Madison, Wis.). The composition of the universal binding buffer was: 10mM Tris-HCl (pH 7.5), 4% glycerol, 1mM MgCl2, 0.5mM EDTA, 5mM DTT, 50mM NaCl, 0.05mg/mL poly (dI-dC) · (dI-dC). The electrophoresis buffer used was 0.5 XTBE (0.9M Tris base, 0.9M boric acid, 0.02M EDTA, pH 8.0). PBST (phosphate buffer, 0.1% Tween 20) was used as a wash. Agarose for electrophoresis was obtained from Biowest (Miami, FL). Bovine Serum Albumin (BSA) was purchased from Amresco (Solon, OH.) Cy 3-labeled streptavidin was purchased from Amersham Biosciences (Uppsala, Sweden).

The experimental steps are as follows:

preparation of DNA probes: the probes were synthesized by Shanghai Boya, and the sequences of the probes are shown in Table 2 (the LFP and LP' probes in each group of probes constitute capture probes for the corresponding transcription factors; the IP probes are immobilized probes). The method for preparing the immobilized probe in each group was the same as that in the section "DNA Probe preparation" in example 1. The protein capture probes were all solubilized with water and the corresponding probes (LFP and LP' probes in each set of probes) were annealed to form duplexes, wherein the final concentration of each set of probes was 60 nanomoles per liter.

B, preparation of DNA chip: the same procedure as in "preparation of DNA chip" in example 1 was repeated.

C. Configuration of nucleic acid-protein binding system: the three transcription factors (1.0. mu.g AP1, 100ng NFkB and 0.1. mu. gSp1) and protein capture probe were mixed and universal binding buffer was added to bring the final concentration of buffer to 1X. The reaction was combined at room temperature for 30 minutes.

D. Isolation of nucleic acid-protein binding System: a2% agarose gel and TBE buffer for electrophoresis were prepared in advance, and the mixture was precooled to 4 ℃. The above-mentioned binding reaction system was added to the well of the gel and electrophoresed at 120 volts for 20 minutes. The desired gel sections were cut out according to the position of the bromophenol blue indicator in the running buffer.

E. Recovery of nucleic acids: the nucleic acid in the gel obtained in the previous step was recovered using QIAGEN type II gel recovery kit from Qiagen, according to the product instructions. Finally, elution was carried out with 20. mu.l of eluent.

F. Hybridization analysis of the chip: and (3) vacuumizing the nucleic acid obtained in the previous step, adding 5 microliters of water for redissolving, adding dNTP and PCR buffer solution, and adding a primer with Cy 3-labeled T7 Pro for nucleic acid amplification. The PCR procedure was: 5 minutes at 95 ℃; repeating 40 cycles of 95 degrees for 30 seconds, 53 degrees for 30 seconds, and 72 degrees for 20 seconds; 7 minutes at 73 degrees. The amplification product was vacuum-dried, redissolved with 5. mu.l of water, and prepared into a 15. mu.l system of a hybridization solution (into which the HC-LP probe was also added here) containing 3XSSC and 0.1% SDS, was applied to a DNA chip and hybridized at 65 ℃ for 1 hour. The slides were then washed with 0.2XSSC and 0.1% SDS wash for 10 minutes at room temperature and spun dry at 1000 rpm. The image was scanned using a Scanarray 4000 scanner and the images were analyzed using GenePix.

The detection of the three proteins of this example was carried out by the "end-labeling method" of example 1 as a control of the detection results of this example.

The detection results are shown in FIG. 6A and FIG. 6B, respectively, and FIG. 6A is a graph showing the results of an experiment in which the "single-strand amplification method" is used for detection; FIG. 6B is a graph showing the results of detection using "end-labeling method" in example 1, in which A1-A5 are AP1 detection results; B1-B5 are NFkB detection result points; C1-C5 are hybridization control points (HC points, HC-IP and HC-LP sequences are shown in Table 1); D1-D5 are Sp1 detection result points. The result shows that the detection by adopting the single-chain amplification method can improve the detection sensitivity of the method, so that the signal points which cannot be detected by adopting the end labeling method can be clearly and sensitively detected.

TABLE 2 Probe sequence Listing with 3' overhang sequences

Group number	TF	Probe numbering	Name of probe	Probe sequence
					1	AP-1	1	AP-1-IP	5′-T20-CGCTTGAT GAGTCAGCCGGA-TCCCG-3′
22	AP-1-LFP	5′-CGCTTGA TGAGTCAGCCGGA-CCCTATAGTGAGTCGTATTACCCC-3′
			26	AP-1-LP’			5′-CGGGA-TCCGGC TGACTTCATCAAGCG-3′
2	NFκB	5						NFκB-IP	5′-T20-AGTTGAG GGGACTTTCCCAGGA-TCCCG-3′
			23	NFκB-LFP	5′-AGTTGAG GGGACTTTCCCAGGA-CCCTATAGTGAGTCGTATTACCCCC-3′
		27				NFκB-LP’	5′-CGGGA-TCCTG GGAAAGTCCCCTCAACT-3′
			3	SP1	9			SP1-IP	5′-T20-AAAGC CCCGCCCCGATATAAT-TCCCG-3′
24	SP1-LFP	5′-AAAGC CCCGCCCCGATATAAT-CCCTATAGTGAGTCGTATTACCCC-3′
					28	SP1-LP’	5′-CGGGA-ATTATATC GGGGCGGGGCTTT-3′
4		25						T7 Pro	5’-Cy3-GGGGTAATACGACTCACTAAGGG-3’

Claims (24)

1. A method for detecting nucleic acid binding protein capable of binding to specific sequence based on biochip includes the following steps: 1) adding a solution containing a plurality of groups of nucleic acid capture probes into a biological sample system containing a plurality of nucleic acid binding proteins to be detected to be capable of binding with specific sequences to form a nucleic acid capture probe-nucleic acid binding protein complex capable of binding with the specific sequences, wherein the nucleic acid sequence of the nucleic acid capture probe contains at least one binding sequence capable of binding with the nucleic acid binding protein capable of binding with the specific sequences; 2) separating the nucleic acid capture probe-nucleic acid binding protein complex capable of binding to the specific sequence, and recovering the nucleic acid capture probe; 3) hybridizing the nucleic acid capture probe recovered in the step 2) with a plurality of single-stranded immobilized probes which are immobilized on a biochip substrate and correspond to the nucleic acid capture probe, wherein the nucleic acid sequence of the immobilized probes is complementary with the nucleic acid capture probe or one strand of the nucleic acid capture probe corresponding to the immobilized probes; 4) and detecting the hybridization result.

2. The method of claim 1, wherein: the separation of the nucleic acid capture probe-nucleic acid binding protein complex capable of binding to the specific sequence in the step 2) is carried out according to the following process: separating the mixed sample obtained in the step 1) by gel electrophoresis, and obtaining the nucleic acid capture probe-nucleic acid binding protein complex capable of binding with the specific sequence by cutting gel, separating by using a kit or using electroelution.

3. The method of claim 1, wherein: the separation of the nucleic acid capture probe-nucleic acid binding protein complex capable of binding to the specific sequence in the step 2) is carried out according to the following process: separating the mixed sample obtained in the step 1) by adopting a chromatographic column method to obtain a nucleic acid capture probe-nucleic acid binding protein compound capable of binding with a specific sequence.

4. The method of claim 1, wherein: the separation of the nucleic acid capture probe-nucleic acid binding protein complex capable of binding to the specific sequence in the step 2) is carried out according to the following process: separating the mixed sample obtained in the step 1) through a filter membrane to obtain a nucleic acid capture probe-nucleic acid binding protein compound capable of binding with a specific sequence, wherein the filter membrane can adsorb protein.

5. The method of claim 1, wherein: the separation of the nucleic acid capture probe-nucleic acid binding protein complex capable of binding to the specific sequence in the step 2) is carried out according to the following process: adding an antibody which can specifically recognize each nucleic acid binding protein capable of binding with a specific sequence into the mixed sample obtained in the step 1), and separating to obtain a nucleic acid capture probe-nucleic acid binding protein complex capable of binding with the specific sequence by using an antibody purification method.

6. The method of claim 1, wherein: the separation of the nucleic acid capture probe-nucleic acid binding protein complex capable of binding to the specific sequence in the step 2) is carried out according to the following process: separating the mixed sample obtained in the step 1) by using capillary electrophoresis, and collecting the nucleic acid capture probe-nucleic acid binding protein complex capable of binding with the specific sequence.

7. The method of claim 1, wherein: one nucleic acid strand of each set of said nucleic acid capture probes has an overhang sequence at one end.

8. The method of claim 7, wherein: the immobilized probe is fully complementary to the nucleic acid strand with the overhang sequence in its corresponding nucleic acid capture probe.

9. The method of claim 8, wherein: the nucleic acid strand of the nucleic acid capture probe with the overhang sequence also carries a label molecule.

10. The method of claim 9, wherein: the labeled molecules are biotin, digoxigenin, fluorescent labels, quantum dots, gold particles or nanoparticles.

11. The method according to any one of claims 1 to 6, wherein: and (3) amplifying the recovered nucleic acid capture probe by using a primer, wherein the primer can be hybridized with the nucleic acid strand with the 3' overhang sequence in the nucleic acid capture probe, before the hybridization in the step 3).

12. The method of claim 11, wherein: the 3 ' overhang sequence is the same in one nucleic acid strand bearing a 3 ' overhang sequence of each set of said nucleic acid capture probes, and the sequence of said primer is fully complementary to said 3 ' overhang sequence.

13. The method of claim 12, wherein: when one primer is used for amplification, the primer is also doped with a labeled molecule.

14. The method of claim 13, wherein: the 5' end of the primer molecule is provided with a marker molecule.

15. The method of claim 14, wherein: the labeled molecules are biotin, digoxigenin, fluorescent labels, quantum dots, gold particles or nanoparticles.

16. The method of claim 11, wherein: the amplification with one primer also incorporates a labeled molecule of nucleotide in the amplification feed.

17. The method of claim 16, wherein: the labeled molecules are biotin, digoxigenin, fluorescent labels, quantum dots, gold particles or nanoparticles.

18. The method according to any one of claims 1 to 6, wherein: each set of said nucleic acid capture probes further comprising a 3 'overhang sequence and a 5' overhang sequence at the 3 'end and the 5' end of one nucleic acid strand, respectively; prior to said hybridizing of step 3), amplifying said recovered nucleic acid capture probe with two primers, one of said two primers being capable of hybridizing to the 3 'overhang of the nucleic acid strand of said nucleic acid capture probe having a 3' overhang sequence and a5 'overhang sequence, and the other primer having a sequence identical to the 5' overhang of the nucleic acid strand of said nucleic acid capture probe having a 3 'overhang sequence and a 5' overhang sequence.

19. The method of claim 18, wherein: the 3 'overhang sequence in the nucleic acid strand with a 3' overhang sequence and a5 'overhang sequence of the nucleic acid capture probe is the same, the 5' overhang sequence in the nucleic acid strand with a 3 'overhang sequence and a 5' overhang sequence of the nucleic acid capture probe is the same, one of the two primers is capable of hybridizing to the 3 'overhang of the nucleic acid strand with a 3' overhang sequence and a5 'overhang sequence in the nucleic acid capture probe, and the sequence of the other primer is identical to the 5' overhang of the nucleic acid strand with a 3 'overhang sequence and a 5' overhang sequence in the nucleic acid capture probe.

20. The method of claim 19, wherein: and when the two primers are used for amplification, the primers are also doped with a labeled molecule.

21. The method of claim 20, wherein: the 5' end of the primer molecule is provided with a marker molecule.

22. The method of claim 21, wherein: the labeled molecules are biotin, digoxigenin, fluorescent labels, quantum dots, gold particles or nanoparticles.

23. The method of claim 18, wherein: the amplification using the two primers also incorporates nucleotides with labeled molecules in the amplification raw material.

24. The method of claim 23, wherein: the labeled molecules are biotin, digoxigenin, fluorescent labels, quantum dots, gold particles or nanoparticles.