Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
  • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

• the present invention relates to a method for detecting a nucleic acid binding protein, and more particularly to a method for detecting a nucleic acid binding protein based on a biochip.
• Nucleic acid binding proteins include double-stranded DNA-binding proteins, single-stranded DNA-binding proteins, and R A binding proteins.
• Double-stranded DNA-binding proteins are a class of protein molecules or protein-molecular complexes that specifically bind to a specific sequence of double-stranded DNA.
• Double-stranded DNA-binding proteins mainly include repressors in prokaryotes, operator proteins, and transcription factors in eukaryotes. These double-stranded DNA-binding proteins activate, inhibit, attenuate or enhance the expression of specific genes by binding to a specific sequence of operator/promoter.
• repressor proteins and operator proteins in prokaryotes are relatively simple, and can regulate the expression of enzymes encoding the metabolic processes of cells, encoding antibiotic resistance genes, and adapt the physiological activities of cells to the external environment; eukaryotes
• the transcription factors are versatile, and various phenomena such as cell cycle, apoptosis, and carcinogenesis are closely related to specific transcription factors.
• the expression of genes encoding proteins is regulated by transcription factor-mediated transcriptional activation, transcription, post-transcriptional modification (including RNA cleavage, capping and tailing).
• transcription, post-translational modifications including phosphorylation, glycosylation, acetylation, etc.
• pre-transcriptional, post-transcriptional, and post-translational are regulated at three levels: pre-transcriptional, post-transcriptional, and post-translational.
• Transcription factor-mediated transcriptional activation is important as the first loop of the entire gene expression regulatory network. Most of the organism's stress response to the external environment is achieved by opening or shutting down specific genes through specific transcription factors. Studies have shown that the expression of most eukaryotic genes is regulated by one or some specific transcription factors. The more complex organisms, the more genes that encode transcription factors, and the more complex the mechanisms of gene expression regulation. 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.
• transcription factors are products of protooncogenes that express or enhance expression only in malignant tumor tissues (such as FOS and C-Myc), while some transcription factors are only weakly expressed in malignant tumor tissues or No expression (such as p53 and E2F). Therefore, if it is possible to detect the expression level of some transcription factors or all transcription factors at a certain moment in the organism, Known transcription factor regulatory gene data, you can get the pre-transcriptional level of regulation information, may be used to diagnose tissue cancer, can also screen drug targets, study some cellular stress response mechanisms, observe the cell's signaling pathway Activate or deactivate, etc.
• cDNA microarray technology can give data on the mRNA expression status of all transcription factor-encoding genes at the genome-wide level.
• active transcription factors can regulate the expression of genes, and the activity of transcription factors is often regulated by various modifications such as phosphorylation, acetylation and glycosylation.
• the localization of transcription factors in cells also affects transcription factors. The activity, so the number of active transcription factors and the expression of mRNA or protein levels of transcription factors are not directly related. For example, in various stages of the cell cycle, the expression of the mRNA and protein levels of the transcription factor Yin Yang 1 (YYl) is almost constant, but the number of active YY1 varies regularly with the cell cycle. Therefore, cDNA microarrays cannot theoretically give transcription factor expression profile data of interest to biologists.
• the conventional "active" double-stranded DNA-binding protein (transcription factor) is detected by EMS A (electrophoretic Mobility Shift Assay, gel shift, band shift).
• EMS A electrophoretic Mobility Shift Assay, gel shift, band shift.
• the sample to be tested is mixed with a known DNA double-stranded molecule with an isotope label, and subjected to polyacrylamide gel electrophoresis under non-denaturing conditions.
• BD Biosciences Clontech (Palo Alto, CA) in the United States has introduced a MercuryTM Transcription Factor Kit.
• the kit gives a 96-well plate for detecting transcription factors. Each hole in the 96-well plate has a strap that can be used with A double-stranded DNA probe of a nucleic acid sequence to which a transcription factor specifically binds.
• the sample to be detected is added to the well for reaction, and the probe binds to the corresponding transcription factor in the sample to be tested. After washing, a primary antibody that specifically recognizes the transcription factor is added, and an enzyme-labeled secondary antibody is added to the primary antibody for chemiluminescence detection.
• DNA-binding proteins There are also sequence-specific single-stranded DNA-binding proteins and RNA-binding proteins in organisms, most of which regulate the physiological activities of organisms.
• antibody-like nucleic acid ligands that specifically bind to target protein molecules can be screened by in vitro evolution ( «Wfro evolution), and these nucleic acid binding proteins do not have corresponding high-throughput analytical methods.
• the method for detecting a nucleic acid binding protein based on a biochip comprises the following steps: 1) adding a solution containing several sets of nucleic acid capture probes to a biological sample system containing a plurality of nucleic acid binding proteins to be detected to form a nucleic acid capture a nucleic acid binding protein complex, the nucleic acid sequence of the nucleic acid capture probe comprising at least one binding sequence capable of binding to a nucleic acid binding protein of interest; 2) isolating the nucleic acid capture probe-nucleic acid binding protein complex, And then recovering the nucleic acid capture probe; 3) hybridizing the nucleic acid capture probe recovered in step 2) to a plurality of single-stranded immobilized probes immobilized on the biochip substrate and corresponding to the nucleic acid capture probe, The nucleic acid sequence of the immobilized probe is complementary to one of the corresponding nucleic acid capture probes or nucleic acid capture probes; 4) the hybridization result is detected.
• the step 2) isolating the nucleic acid capture probe-nucleic acid binding protein complex by the following five processes: a) separating the mixed sample obtained in the step 1) by conventional gel electrophoresis, and cutting the gel with a reagent The nucleic acid capture probe-nucleic acid binding protein complex is obtained by separation or electroelution. b) The mixed sample obtained in the step 1) is separated by a chromatographic method to obtain a nucleic acid capture probe-nucleic acid binding protein complex. c) The mixed sample obtained in the step 1) is passed through a filter to obtain a nucleic acid capture probe-nucleic acid binding protein complex, and the filter can adsorb the protein.
• step 1) in the mixed sample obtained in step 1) An antibody that specifically recognizes each nucleic acid binding protein is added, and a nucleic acid capture probe-nucleic acid binding protein complex is isolated using a method similar to antibody purification (for example, using a protein A/G-coated agarose beads-bound antibody).
• step 1) The mixed sample obtained in the step 1) is separated by a capillary electrophoresis apparatus, and the nucleic acid capture probe-nucleic acid binding protein complex is automatically collected.
• the method of the present invention can be named "end labeling method" and can be used to detect nucleic acid binding proteins, such as double-stranded DNA binding proteins, single-stranded DNA binding proteins, RNA-binding proteins (such as HuB, HuC, ELAV, etc.), etc., by in vitro evolution.
• nucleic acid binding proteins such as double-stranded DNA binding proteins, single-stranded DNA binding proteins, RNA-binding proteins (such as HuB, HuC, ELAV, etc.), etc.
• the non-natural nucleic acid ligands such as the aptamer of thrombin
• aptamer of thrombin which can be bound by the method can also be detected by the method of the present invention.
• the nucleic acid binding protein is preferably a double-stranded DNA-binding protein, more preferably a transcription factor, tU API, Spl, p53, E2F or the like.
• the nucleic acid sequence of the nucleic acid capture probe contains a binding sequence capable of binding to a nucleic acid binding protein of interest; a nucleic acid strand of each of the nucleic acid capture probes One end has a sequence of protruding ends.
• the immobilized probe is completely complementary to the nucleic acid strand with the overhang sequence in its corresponding nucleic acid capture probe.
• one end of the nucleic acid strand with the overhang sequence in the nucleic acid capture probe further carries a label molecule, wherein, preferably, biotin, digoxin, fluorescent label, quantum dot, gold particle , nanoparticles.
• each of the nucleic acid capture probes has a 3' end with a 3' end at the 3' end of the nucleic acid strand.
• the nucleic acid capture probe is further amplified by a primer, and the primer is capable of interacting with the nucleic acid strand having the 3' overhang sequence in the nucleic acid capture probe. Hybridization is facilitated for subsequent nucleic acid amplification steps.
• the 3' overhang sequence in a nucleic acid strand having a 3' overhang sequence of each set of said nucleic acid capture probes is identical, the sequence of said primer and said 3' overhang The end sequences are fully complementary.
• a labeling molecule may be added to the system, and the labeling molecule may be first amplified with a labeling molecule, or a labeled molecule may be incorporated in the amplification material during amplification.
• double-strand amplification method before hybridization the 3' end and the 5' end of each nucleic acid strand of each group of the nucleic acid capture probes are respectively Carrying a 3' overhang sequence and a 5' overhang sequence; performing the step 3) before the hybridization, using the two primers to amplify the recovered nucleic acid capture probe, one of the two primers Hybridizing to the 3' overhang of the nucleic acid strand with the 3' overhang sequence and the 5' overhang sequence in the nucleic acid capture probe, the sequence of the other primer and the 3 in the nucleic acid capture probe
• the 'end-end sequence is identical to the 5' overhang of the nucleic acid strand of the 5' overhang sequence.
• the 3' overhang sequence in the nucleic acid strand with the 3' overhang sequence and the 5' overhang of the nucleic acid capture probe is the same, and the nucleic acid capture probe has 3 The 5' overhang sequences in the nucleic acid strand of the 'overhanging sequence and the 5' overhang are identical, and the two primers are capable of binding to the nucleic acid capture probe with a 3' overhang sequence and a 5' overhang.
• the 3' overhang of the nucleic acid strand of the end sequence hybridizes, and the sequence of the other primer coincides with the 5' overhang of the nucleic acid strand with the 3' overhang sequence and the 5' overhang sequence in the nucleic acid capture probe.
• the operation procedure of detection by double-strand amplification method is shown in Fig. 7.
• the capture probe 1 is mixed with the target protein 3 (circular and triangular in the figure), and the nucleic acid capture probe-nucleic acid binding protein is obtained after separation.
• Complex 4 recovering the capture probe; the recovered capture probe is amplified by two primers 6, and the amplified product is hybridized with the chip 5 immobilized with the immobilized probe 2, and the hybridization signal is detected to be detected. result.
• the capture probe contains a binding sequence for the protein of interest, one of the two strands with a 3' overhang and 5, overhang
• the sequence of the ligature 1 is complementary to the 3' overhang sequence, and the sequence of the stalk 2 is identical to the 5' overhang; the immobilized probe sequence is identical to the binding sequence.
• a labeling molecule can be added to the system, and the labeling molecule can be first amplified with a labeling molecule, or a labeled molecule can be incorporated in the amplification material during amplification.
• Nucleotides, wherein the labeling molecules are preferably biotin, digoxigenin, fluorescent label, quantum dots, gold particles, nanoparticles.
• Figure 1 is a schematic view showing the operation of the invention for detecting a plurality of nucleic acid binding proteins using "end labeling";
• Figure 2 is a diagram showing the probe structure of a plurality of nucleic acid binding proteins detected by "end labeling method" of the present invention
• 3A is a dot matrix design diagram of the embodiment 1;
• Figure 3B is an experimental result of simultaneous detection of three binding proteins in Example 1;
• Fig. 3C is the experimental result of simultaneous detection of three binding proteins in Example 1, in which a competitive binding probe of API is added to the reaction system;
• Figure 3D shows the results of simultaneous detection of three binding proteins in Example 1, in which a competitive binding probe of NFkB was added to the reaction system;
• Figure 3E shows the results of the simultaneous detection of three binding proteins in Example 1, in which a competitive binding probe of Spl was added to the reaction system;
• Figure 4 is a schematic view showing the operation flow of detecting a plurality of nucleic acid binding proteins using "single-strand amplification method" of the present invention
• Figure 5 is a schematic view showing the structure of a probe and a primer for detecting a plurality of nucleic acid binding proteins using "single-strand amplification method";
• 6A is an experimental result of simultaneous detection of three binding proteins (AP1, NFkB, Spl) using "single-strand amplification method" in Example 2;
• Figure 6B is an experimental result of the second embodiment using the same "end labeling method" as in Example 1;
• Figure 7 is a schematic view showing the operation flow of detecting a plurality of nucleic acid binding proteins using "double-strand amplification method" of the present invention.
• Fig. 8 is a schematic view showing the structure of a probe and a primer for detecting a plurality of nucleic acid binding proteins by the method of chain amplification according to the present invention.
• Example 1 Simultaneous detection of three nucleic acid binding protein APIs, NFkB and Spl (end labeling method) using a DNA chip
• the operation procedure using the end labeling method is shown in Fig. 1.
• the capture probe 1 is mixed with the target protein 3 (circle and triangle in the figure), and the nucleic acid capture probe-nucleic acid binding protein complex is obtained after separation. 4.
• the capture probe is recovered, and then hybridized with the chip 5 to which the immobilized probe 2 is immobilized, and the hybridization signal is detected to obtain a detection result.
• the preferred probe structure of the capture probe and the immobilized probe used is as shown in FIG. 2:
• the capture probe contains a binding sequence of a protein of interest, and one of the two strands has an overhang and a label.
• biotin is used; the immobilized probe is complementary to the long chain in the capture probe.
• Test Materials - Transcription Factors AP-1 C-Jun (#E3061), NFkB (p50) (#E3770) and Spl (#E6391) from Promega (Madison, Wl).
• the composition of the universal binding buffer is: 10 raM Tris-HC1 (pH 7. 5), 4% glycerol, 1 mM MgC12, 0.5 mM EDTA, 5 mM DTT, 50 mM NaCl, 0.05 mg/mL poly (dl - dC) ⁇ (dl-dC)
• the running buffer used was 0. 5xTBE (0. 9 M Tris base, 0.9 M boric acid, 0.02 M EDTA, pH 8. 0).
• PBST phosphate buffer, 0.1% Tween 20
• Electrophoresis agarose was obtained from Biowest (Miami, FL).
• Bovine serum albumin (BSA) was purchased from Amresco (Solon, OH).
• Cy3-labeled streptavidin was purchased from Amersham Biosciences (Uppsala, Sweden).
• A. Preparation of DNA probes All probes were synthesized by Shanghai Boya Company. The sequence of the probes is shown in Table 1. (AP- 1-IP is a fixed probe of API, AP- 1- LP, AP- 1- FP consists of a capture probe of API, AP-1-CP and AP-1-FP constitute a competitive binding probe of API, and the expressions of other probes are similar; the protruding end of LP probe of this embodiment The sequence is 5'-CGGGA-3'. The fixed probes in each group were first dissolved in water and placed in a solution of 50% dimethyl sulfoxide (DMS0) with a final concentration of 10 micromoles per liter.
• DMS0 dimethyl sulfoxide
• the protein capture probes were all dissolved in water and the corresponding probes (FP and LP probes in each set of probes) were annealed to form a double strand with a final concentration of 60 nanomoles per liter for each set of probes.
• the CA) robots placed the above fixed probes onto the amino-modified slides in the dot pattern of Figure 3A at a dot pitch of 350 microns. After the spotting is completed, the slides are baked in an oven at 80 ° C for 1 hour, and then glued at 250 mJ of energy to fix the nucleic acid molecules on the surface of the slide using an ultraviolet glue.
• A1-A5 are API immobilized probes
• Bl-B5 is NFkB fixed probes
• C1-C5 is Spl fixed probes
• D1-D5 is TFIID fixed probes
• E1-E5 is NC fixed probes
• Fl- F5 is a HC immobilized probe
• A6, B6, C6, D6, E6, and F6 are spotted controls.
• the spotted control is a nucleic acid molecule with fluorescein itself. After scanning, the slide can be detected. To the spotted control, the steps to confirm the slide are correct.
• C. Configuration of nucleic acid-protein binding system Mix three transcription factors (1.0 g Fc, 300 ng NFkB and 1.0 ⁇ ⁇ Spl) and protein capture probes (that is, annealing products of LP and FP), and add them to the universal Binding buffer to bring the final concentration of buffer to IX. The reaction system was combined for 30 minutes at room temperature. When the experiments described in Fig. 3C, Fig. 3D, and Fig. 3E were carried out, a competitive binding probe (CP) was further added to the reaction system.
• CP competitive binding probe
• nucleic acid-protein binding system 2% agarose gel and TBE buffer for electrophoresis were pre-configured and pre-cooled to 4 °C. The above binding reaction system was added to the well of the gel and electrophoresed at 120 volts for 20 minutes. The desired gel fraction is cut out based on the position of the bromophenol blue indicator in the running buffer.
• the hybridization analysis of the chip the nucleic acid obtained in the above step is configured into a 15 ⁇ l system of the hybridization solution (the hybridization solution is further provided with an HC-LP probe), which contains 3XSSC and 0.1% SDS, added On the DNA chip, hybridize at 65 1 for 1 hour. Then, the slides were washed with 0. 2XSSC and 0.1% SDS cleaning solution, and dried at 1000 rpm for 10 minutes at room temperature. 1% BSA was added to the chip for blocking, and the reaction conditions were 37 ⁇ 30 minutes. The slides were washed with PBST, room temperature for 10 minutes, and then 1000 rpm for dry slides.
• FIG. 3A is a dot matrix design of the chip
• FIG. 3B is an experimental result of simultaneously detecting three nucleic acid binding proteins on the chip using "end labeling method”
• FIG. 3C is a simultaneous detection of three kinds of chips on the chip using "end labeling method”.
• FIG. 3D is the experimental result of simultaneously detecting the three nucleic acid binding proteins on the chip by using the "end labeling method", and the binding reaction system A competitive binding probe for the nucleic acid binding protein NFkB was added
• Figure 3E is the result of simultaneous detection of three nucleic acid binding proteins on the chip using "end labeling", and the competition of the nucleic acid binding protein Spl was added to the binding reaction system. Bind the probe.
• the three terminal transcription factors can be clearly detected by the "end labeling method" of the present invention, and the sequence in the NC is derived from the promoter sequence of the prokaryotic phage, and the transcription factor binding sequence of the eukaryote is greatly different. , can not bind to any of the above transcription factors, so the signal of the NC probe is always negative; HC is used as a hybridization control, added before hybridization, to normalize different lattices, so the HC signal is always positive .
• the operation flow using the single-strand amplification method is shown in Fig. 4.
• the capture probe 1 is mixed with the target protein 3 (circle and triangle in the figure), and the nucleic acid capture probe-nucleic acid binding protein is obtained after separation.
• the complex 4 the capture probe is recovered; the recovered capture probe is amplified by the primer 6, and the amplified product is hybridized with the chip 5 to which the immobilized probe 2 is immobilized, and the hybridization signal is detected to obtain a detection result.
• the preferred design principle of the capture probe, the immobilized probe and the primer used is as shown in FIG. 5:
• the capture probe contains a binding sequence of a protein of interest, and one of the two strands has a 3'overhang;
• the primer sequence is complementary to the 3'overhang;
• the immobilized probe is identical to the binding sequence.
• the transcription factors AP- l (c-Jun) (#E3061), NFkB (p50) (#E3770) and Spl (#E6391) were from Promega (Madison, WI).
• the composition of the universal binding buffer is: lO mM Tris-HC1 (pH 7.5), 4% glycerol, 1 mM MgC12, 0.5 mM EDTA, 5 mM DTT, 50 mM NaCl, 0.05 mg/mL poly (dl -dC) ⁇ (dl-dC).
• the electrophoresis buffer used was 0. 5xTBE (0. 9 M Tris base, 0.9 M boronic acid, 0.02 M EDTA, pH 8. 0).
• PBST phosphate buffer, 0.1% Tween 20
• Electrophoresis agarose was obtained from Biowest (Miami, FL).
• Bovine serum albumin (BSA) was purchased from Amresco (Solon, 0H).
• Cy3-labeled streptavidin was purchased from Amersham Biosciences (Uppsala, Sweden).
• A. Preparation of DNA probes The probes were synthesized by Shanghai Boya Company. The sequence of the probes is shown in Table 2 (LFP and LP' probes in each set of probes are composed of capture probes corresponding to transcription factors; IP probe The needle is a fixed probe). The fixed probe preparation method in each group was the same as the "DNA probe preparation" portion in Example 1. The protein capture probes were all dissolved in water and the corresponding probes (LFP and LP' probes in each set of probes) were annealed to form a double strand with a final concentration of 60 nanomoles per liter for each set of probes.
• C Configuration of nucleic acid-protein binding system: Mix three transcription factors (1. 0 ⁇ ⁇ ⁇ 1, 100ng FkB and 0.1g Spl) with protein capture probes, and add universal binding buffer to achieve the final concentration of buffer. IX. The reaction system was combined for 30 minutes at room temperature.
• nucleic acid-protein binding system 2% agarose gel and TBE buffer for electrophoresis were pre-configured and pre-cooled to 4 °C. The above binding reaction system was added to the well of the gel and electrophoresed at 120 volts for 20 minutes. The desired gel fraction is cut out based on the position of the bromophenol blue indicator in the running buffer.
• Hybridization analysis of the chip The nucleic acid obtained in the previous step was vacuum-dried, re-dissolved by adding 5 ⁇ l of water, added to dNTP and PCR buffer, and then amplified by primers with Cy3-labeled T7 Pro.
• the PCR procedure is: 95 degrees 5 minutes; 95 degrees 30 seconds, 53 degrees 30 seconds, 72 degrees 20 Seconds, repeat 40 cycles; 73 degrees 7 minutes.
• the smear of the SDS which contains 3XSSC and 0.1% SDS, which is then re-dissolved in 5 ⁇ l of water, and is placed in a 15 ⁇ l system of the hybridization solution (HC-LP probe is also added to the hybridization solution at this time).
• the detection of the three proteins of this example was carried out by using the "end labeling method" of Example 1, as a control for the detection results of the present example.
• FIG. 6A is a diagram showing experimental results of detection by "single-strand amplification method"
• FIG. 6B is a result diagram of detection by using "end labeling method” of Example 1.
• Al-A5 is the API detection result point
• B1-B5 is the NFkB detection result point
• C1-C5 is the hybridization control point (HC point, HC-IP and HC-LP sequence are shown in Table 1)
• D1-D5 is Spr detection Result point.
• the results show that the detection by the single-strand amplification method can improve the detection sensitivity of the method of the present invention, and the signal points which cannot be detected by the terminal labeling method can be clearly and sensitively detected.
• Table 2 Probe Sequence Listing with 3' Overhang Sequence
• the invention adopts a biochip method for detecting nucleic acid binding proteins, especially transcription factors,
• the detection of hybridization signals by the capture probe and the immobilized probe has the advantages of high detection sensitivity and high detection flux; moreover, when using the two improved methods of the present invention (pre-hybridization single-strand amplification, pre-hybridization double-strand When amplifying), the primers can be pre-labeled, and it is no longer necessary to perform molecular labeling modification on each group of nucleic acid capture probes, which can greatly reduce the experimental cost.
• the method of the invention can be widely used in the fields of disease diagnosis, drug target screening, disease mechanism and the like.

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