KR20050106759A - Method for identification and analysis of certain molecules using the dual function of single strand nucleic acid - Google Patents

Abstract

The present invention relates to a method for identification and analysis of a specific substance using a single stranded nucleic acid, and more particularly, to capture an array of capture probes on a solid substrate to prepare an array, and to prepare a specific substance-target probe complex by treatment of analytical reagents. Thereafter, the specific substance-target probe complex is washed and separated according to affinity, and the hybridization solution is added to dissociate the target probe, and then the capture probe and the labeled target probe are reacted, and then the substance labeled on the target probe. The present invention relates to a method for identifying and quantifying a specific substance using a single-stranded nucleic acid array. Specific substance-target probe complex of the present invention is determined the degree of binding according to the specificity and binding strength between them from this it is possible to identify the specific substance and confirm the quantitative change. Accordingly, the present invention provides a very useful method for searching and analyzing several kinds of specific substances in a sample at the same time using a single stranded nucleic acid array and a target probe.

Description

Method for identification and analysis of certain molecules using the dual function of single strand nucleic acid}

The present invention relates to a method for the identification and quantitative analysis of a specific substance, and more particularly, a single-stranded nucleic acid, which is an aptamer, forms a complex with a specific substance or forms complementary single-stranded and double-stranded nucleic acids. It relates to a simple and novel method of searching for and analyzing the identification and quantitative changes of specific substances using dual functions.

The technology to search for and analyze the identification and quantitative change of a specific substance has been developed due to the development of physics and biochemistry, but the problems related to the use and maintenance cost, ease, accuracy, sensitivity, inspection time and process automation of existing methods or devices The need for efficient new methods and devices is very high. Analyzing specific substances is not an ultimate goal in itself, but rather a part of the whole process, which can be useful for microbial and viral identification, cell, protein, and organic material analysis, leading to medical, veterinary, environmental and food engineering. It is widely applied to agriculture and agriculture.

Nucleic acids are linear multimers in which nucleotides are covalently linked, and nucleotides are small organic compounds that are phosphoric acid, sugars and purines (adenine or guanine) or pyrimidines (cytosine, thymidine, uracil). Nucleic acids exist in single- or double-stranded strands, which bind together by hydrogen bonds and interactions between nucleotides under specific physical conditions to form unique conformations, which are determined by single-stranded nucleotide sequences. . In general, nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are stores of information for expressing proteins with cell structure and enzyme activity, but in 1982, RNA forms a specific structure. Since the publication of its activity, there have been many reports on the structural properties of a nucleic acid and its specific function. Nucleic acid is composed of four base repeats to maintain a high diversity to form a large number of three-dimensional structure, these three-dimensional structure is stabilized by interacting with a specific material to form a complex.

Nucleic acid can act as a ligand to a specific substance containing a protein, with high binding capacity and specificity through a constant screening process and sequencing from a library of single stranded nucleic acids arranged in various nucleotide sequences. Nucleic acids that bind to specific substances are being screened. The method of selecting nucleic acid binding to a specific substance is called SELEX (Systematic Evolution of Ligand by Exponential enrichment), and the selected product nucleic acid is called aptamer (Tuerk C. and Gold L .; Science , 249 , pp505-510, 1990), SELEX selects molecules that can bind to nucleic acids in nature as well as to other biomolecules, including proteins that do not bind nucleic acids, with very high affinity. have. Techniques for identifying and analyzing specific substances based on the single-stranded nucleic acids thus selected include molecular beacons (Molecular beacon, Li JJ et al .; Biochem Biophys Res Commun., 292 (1) , pp31-40, 2002), Calorimetric method, Stojanovic MN, Landry DW . ; J. AM. CHEM. SOC., 124 , pp9678-9679, 2002), electrochemiluminescence and enzymatic method, Bruno JG, Kiel JL .; Biotechniques 32 (1) , pp178-80, pp182-3, 2002), but there are no specific techniques for identifying and analyzing specific substances based on the array method.

Identification and analysis of specific substances are frequently performed using physical and chemical properties, and analysis using specificity and affinity between materials is usually performed by methods developed based on immunological methods. An immunological method is a technique in which an antibody binds to a specific antigen in a sample to form a complex according to an antigen-antibody reaction, and then the antigen-antibody complex is analyzed using a labeled secondary antibody. For example, ELISA is a method of analyzing a specific antigen by immobilizing a primary antibody in a solid medium and reacting with a sample containing an antigen, followed by processing a labeled secondary antibody that recognizes the complex. When the labeled secondary antibody recognizes and binds to the primary antibody-antigen complex immobilized on the solid medium, the label binds to the solid medium. Probes used in ELISA are labeled with a fluorescent indicator, dioxygenin, horseradish peroxidase, alkaline phosphatase and the like.

Recently, protein chips have been developed and used to analyze proteins at high speed. Since the protein chip knows the arrangement of the antibodies, it is possible to search and analyze the classification and quantification of specific substances. Protein chip manufacturing method is a method of fixing by spotting (spotting) the antibody using a microarrayer (Microarrayer). Protein chip detection technology needs to be able to detect very weak signals that occur when protein chips are packed with high density of antibodies in a small area to provide as much information as possible on a single chip. In addition, as the bioinformation on proteins increases, the degree of integration is increasing. Accordingly, a new method for detecting quantitatively and quickly and accurately is required. Until now, the detection method is mainly used laser induced fluorescence (laser induced fluorescence) and the electrochemical detection method has been developed. As described above, methods for searching and analyzing the identification and quantification of specific proteins using protein chips have been developed, but there are problems such as using expensive instruments and reagents and performing complicated processes.

Accordingly, the present inventors reacted and washed a target probe, which is an aptamer, and a sample, and performed a reaction and washing process of a specific material-target probe complex on a glass slide to which a single-stranded nucleic acid was attached, followed by laser-induced fluorescence. The present invention has been completed by identifying identified and quantitative changes.

An object of the present invention is to identify and quantitatively change a specific substance by using a dual function of aptamer single stranded nucleic acid complexing with a specific substance in a given environment or forming complementary single stranded and double stranded nucleic acid. By identifying these, it is intended to provide a system for searching and analyzing various biomolecules including microorganisms, cells, and proteins using these.

In order to achieve the above object, the present invention provides a method for fabricating an array by fixing capture probes, which are single-stranded nucleic acids composed of (X) -spacer site (R) -target probe recognition site (V), onto a solid substrate. Stage 1; (Ii) a second step of preparing the specific substance-target probe complex by reacting the specific substance with the target probe by treatment of the assay reagent; (Iii) a third step of washing and separating the specific material-target probe complex prepared in the second step; (Iv) adding a hybridization solution to the separated complex and treating it for 5 minutes at 95 ° C. to dissociate the target probe; (Iii) a fifth step of reacting the capture probe with the labeled target probe; And (iii) provides a method for identifying and quantifying a specific material using a single-stranded nucleic acid array consisting of a sixth step of searching for the material labeled on the target probe.

The specific substance may be at least one selected from the group consisting of bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies and enzymes.

The present invention also includes an array made of a capture probe, which is a single-stranded nucleic acid based on the nucleotide sequence of the single-stranded nucleic acid, which is an aptamer for Escherichia coli, and a single-stranded nucleic acid or 0.2% BSA, which is used in the reaction of the target probe and E. coli. It provides an assay kit for the identification and quantitative analysis of E. coli, including the assay reagent, hybridization solution, Cy-5 attached 5'-primer, 3'-primer.

Hereinafter, the present invention will be described in more detail.

The present invention provides a method for manufacturing an array comprising: (i) securing a capture probe, which is a single-stranded nucleic acid composed of a reactor (X) -spacer site (R) -target probe recognition site (V), onto a solid substrate; (Ii) a second step of preparing the specific substance-target probe complex by reacting the specific substance with the target probe by treatment of the assay reagent; (Iii) a third step of washing and separating the specific material-target probe complex prepared in the second step; (Iii) a fourth step of dissociating the target probe by adding a hybridization solution to the separated complex and treating it for 5 minutes at 95 ° C. (iii) a fifth step of reacting the labeled probe with the labeled target probe; And (iii) provides a method for identifying and quantifying a specific material using a single-stranded nucleic acid array consisting of a sixth step of searching for the material labeled on the target probe. As shown in FIG. 1, the single-stranded nucleic acid, which is an aptamer, forms a complex with a specific substance in the Select buffer, or forms a complementary single-stranded nucleic acid and a double-stranded nucleic acid in a nucleic acid hybridization solution. Identification and political analysis of specific substances used.

The first step of the present invention is to prepare an array to which capture probes, which are single stranded nucleic acids, are attached. Specifically, the present invention provides a single-stranded nucleic acid consisting of three parts, which is a capture probe to be fixed to a single-stranded nucleic acid array, and a reactor (X) -spacer site (R) -target probe recognition site (V). do.

The capture probe has a great influence on the degree of hybridization, and its sequencing is a very important step. Each probe constituting the array consists of a characteristic base so that the conjugates must maintain appropriate Tm values. Accordingly, the degree of hybridization of the conjugate should be able to maintain its signal value that is not contaminated by other target probes labeled with fluorescence.

The target probe recognition site (V) is based on the nucleotide sequence of the single-stranded nucleic acid selected by SELEX, preferably, is a single-stranded nucleic acid complementary to the target probe containing a site that recognizes a specific substance, 50 to 100bp As a single-stranded nucleic acid having a nucleotide sequence, the inventors of the aptamer (aptamer) is a single-stranded nucleic acid selected by SELEX standard method (Bock LC et al .; Nature , 355 (6360), pp564-6, 1992) It includes the nucleotide sequence analyzed by separating the single-stranded nucleic acid that specifically binds to E. coli by the (Korean Patent Application No. 2002-0064027).

The base sequence of the spacer region (R) may basically design or jointly use one base sequence for each specific substance to be identified. Considerations in the design of a real spacer include the length of the sequence, base composition, inclusion of non-complementary sequences, and self-conplementarity. The spacer site is a site in consideration of a problem occurring when attached to the substrate surface, the spacer includes a single stranded nucleic acid having a base sequence of 10 to 30bp and a peptide having a 5 to 20 amino acid sequence, preferred embodiments of the present invention For example, the nucleotide sequence of SEQ ID NO: 1 is provided.

In addition, the reactor (X) portion is a site in which a reactor for attaching single-stranded nucleic acid to the substrate of the array is bonded, the reactor may be used amine (amine) group, thiol (-SH) group, biotin (biotin) and the like. .

Preferred single-stranded nucleic acid capture probes of the present invention include a 5'-primer (SEQ ID NO: 1), a single-stranded nucleic acid sequence of SEQ ID NOS: 3 to 26, and a 3'-primer (SEQ ID NO: 2) wherein 5 'of the nucleic acid is modified with an amine group. It is composed of a single-stranded nucleic acid, which is prepared by separating the single-stranded nucleic acid specifically binding to Escherichia coli among the single-stranded nucleic acids selected by SELEX standard method, and then repeatedly performing PCR with primers of SEQ ID NO: 1 and SEQ ID NO: 2 Can be.

In addition, the substrate used in the production of the single-stranded nucleic acid array of the present invention may be an inorganic material such as glass or silicon, or a polymer such as acrylic or polyethylene terephtalate (PET), polycarbonate, polystyrene, polypropylene, or the like. It is made of a material, preferably a slide glass made of glass. At this time, the substrate is coated with an aldehyde or poly L- lysine (lysine) and the like.

More specifically, the capture probe to be immobilized on the surface of the glass slide comprises a single stranded nucleic acid cloned plasmid selected from the SELEX standard method as a template, and the 5'-primer and 5 'side modified with an amine group. Prepare by performing PCR with '-primer. Unbound primers are removed with a PCR product purification kit and nucleic acid, which is a capture probe, to be immobilized on a slide is prepared. The glass slide uses an aldehyde plated slide to produce an array of single stranded nucleic acids as shown in FIG. 6. The fabrication of the array uses a pin-type microarrayer system (GenPak), and spot spacing allows the center-to-center to be 350-400 μm. . Each single-stranded nucleic acid is dissolved in 3 μS SSC buffer to adjust the concentration. At this time, the humidity in the arrayer is maintained at 70 to 80% to perform spotting. The spotted slides are left in a humidified chamber and then baked at 80 ° C. for 2 to 4 hours to perform baking. After the single-stranded nucleic acid is fixed to the glass slide by a method known in the art, the slide is immediately centrifuged and dried, and then stored in a light-blocked state until use.

An array of single-stranded nucleic acids (Fig. 1) can be prepared by various methods known in the art (M. schena; DNA microarray; a practical approach, Oxford, 1999). same.

The second step of the present invention is a step of preparing a specific substance-target probe complex by reacting a specific substance and a target probe by the treatment of the assay reagent. In this case, the target probe may be prepared by labeling with a single stranded nucleic acid, which is an aptamer that binds to a specific substance, and may be prepared by labeling a single stranded nucleic acid that is complementary to the nucleotide sequence of the single stranded nucleic acid, which is an aptamer. Different types of labeling materials can be used in order to recognize signals smoothly. In the former case, the target probe is a single-stranded nucleic acid selected from SELEX or a single-stranded nucleic acid having a structure capable of specific binding with a target substance. For this purpose, the secondary probe is formed by using the MFOLD program that models the secondary structure of the nucleic acid. And after confirming the structure free energy, single-stranded nucleic acids having the most stable secondary structure are selected.

As a label of the target probe, a radioactive isotope, a fluorescein (Fluorescein), a fluorescent substance such as Cy3 or Cy5, or a biotin may be used, and preferably, a fluorescent substance is used. The specific substance is from a group consisting of bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies and enzymes isolated therefrom. In at least one selected, said cell lines and tissues include those derived from prokaryotes or eukaryotes, and eukaryotes refer to humans, animals and plants.

Specific details of the reaction step is that when the target probe is prepared by labeling with a single stranded nucleic acid, which is an aptamer binding to a specific substance, the analysis sequence is shown in FIG. 4A and the single stranded nucleic acid complementary to the nucleotide sequence of the aptamer single stranded nucleic acid. In the case of preparing by labeling, the analysis procedure is shown in FIG. 4B, and the former is named as an aptamer probe method and the latter as an antiaptamer probe method.

In this case, the aptamer probe method is a reaction between a specific substance and a target probe which is a labeled aptamer, and the anti-aptammer probe method is a reaction between a specific substance and an unlabeled aptamer acid. Composition of the salt to which it can bind, an element which prevents nonspecific binding, and the like.

 The assay reagent comprises (i) 20 to 100 mM, preferably 50 mM of tris hydrochloric acid (pH 7.4), (ii) a substance involved in the secondary structure stabilization of single-stranded nucleic acids for buffering against pH. Containing potassium chloride, sodium chloride, and magnesium chloride at 0 to 200 mM, preferably 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, (iii) 0.05 to 0.2% sodium azide, and preferably 0.1 A nucleic acid containing% sodium azide and (iii) inhibiting 0.1% to 0.3%, preferably 0.2% of bovine serum albumin or nonspecific binding for nonspecific binding inhibition, which is a nucleic acid determined in consideration of the specificity of the probe. The assay reagent is a reagent comprising three or more of the four basic components mentioned above. The reaction temperature is ideally carried out at a temperature lower than the SELEX operating temperature, preferably the reaction is carried out at a temperature of 20 to 37 ℃. In general, the protein and single-stranded nucleic acid is excessively treated to prevent nonspecific binding of the target probe, and preferably the yeast tRNA, salmon sperm DNA or human placental DNA of the target probe. DNA) can be used.

The third step of the present invention is to remove the target probe having a low affinity from the specific substance-target probe complex, and washing and separating the specific substance-target probe complex prepared above. This can be separated by membrane filtration, adsorption or centrifugation. When the specific material sample used in the second step is a microorganism and a cell, the target probe solution is mixed with a selection buffer containing the sample and reacted at room temperature or 37 ° C. for 30 minutes, and then immediately filtered by centrifugation or 0.42 μm membrane. In the method, the complex may be washed with various concentrations of Selex buffer, distilled water, or 0.05M EDTA, and then used for secondary reaction by removing the target probe according to the unbound target probe and the binding force. At this time, the washing buffer is preferably 0 to 1 x Selex buffer or 0 to 500 mM EDTA solution. However, if the specific material sample is a protein, the filtration method using a 0.45 μm nitrocellulose membrane filter, a protein labeling method, or a protein sample is adsorbed on an ELISA plate. It can also separate by adsorption method.

In the fourth step of the present invention, the hybridization solution is added to the separated specific substance-target probe complex and heated at 95 ° C. for 5 minutes to dissociate the complex into the specific substance and target probe.

The aptamer probe method uses the dissociated specific substance-target probe complex in the fifth step, and the anti-aptammer probe method labels the single stranded nucleic acid complementary to the single stranded nucleic acid which is the aptamer in the supernatant separated by the above-described principle separation method. After the addition, the reaction was carried out for 0 to 30 minutes, and then the reaction product was used in the fifth step. The fifth step reaction is a step of prehybridizing the array to which the capture probe, which is a single stranded nucleic acid, is attached in a hybridization solution, and then reacting the array by treating the fourth step reaction product, followed by washing and drying.

The fifth step of the present invention is a step of reacting the capture probe with the labeled target probe. Most important at this stage is the hybridization rate and the stability of the hybrid. External factors affecting them include temperature, ionic strength, formamide and other polymers.Intrinsic factors include DNA length, sequence complexity, and mixed state. If mixed phase hybridization is a fixed DNA environment.

In the identification and quantitative analysis of biomolecules using the single-stranded nucleic acid-attached array of the present invention, a nucleic acid hybridization solution for specific binding between a single-stranded nucleic acid capture probe and a target probe is provided. In this case, the hybridization solution is (i) 0.5 to 1.5 M sodium chloride, (ii) 0 to 1.0 M sodium citrate, (iii) 0.1 to 0.3% bovine serum albumin, which is a substance that inhibits nonspecific binding, 0 to 1.0% SDS , 0-10 times Denhart solution or 0-1% nonfat dried milk, (iii) nucleic acid that inhibits nonspecific binding, (v) 30-60% (v / v ) Formamide. Preferably, 1 M sodium chloride, 0.3 M sodium citrate, 0.5% SDS or 100 ug / ml salmon sperm DNA, 0.2% bovine serum albumin or single stranded nucleic acid. After hybridization is completed, the array is washed with 0.11X SSC, 0.2% SDS, etc. at 22-70 ° C. for 30 minutes, washed again with distilled water for 30 seconds, and then dried under dark conditions. The first wash solution is most important when optimizing the wash conditions, so use several washes at this stage to adjust the stringency. Examples of the composition of the washing solution include 1X SSC + 0.2% SDS, 1X SSC + 0.2% SDS, 0.5X SSC + 0.2% SDS, 0.01X SSC + 0.2% SDS.

Specific substance-target probe complex or target probe-capture probe duplex structure is determined by the specificity and binding strength between them, from which specific substances can be identified and quantified. The specificity and binding capacity of the aptamer single-stranded nucleic acid and the specific substance change the binding degree of the complex and determine the type and amount of the specific-single-nucleic acid complex. The single-stranded nucleic acid, or target probe, dissociated from the complex on the array affects the type and amount of the target probe-capture probe double structure and determines the degree of fluorescence generated in the formed double structure. Therefore, by analyzing the degree of fluorescence it is possible to confirm the type and quantitative change of a specific material.

The sixth step of the present invention is to search for a substance labeled on the target probe, and various methods such as FIGS. 5 and 6 may be selected according to the type of the substance labeled on the target probe. 5 shows that after placing the array 7 on the plate 6 , the light irradiated from the lamp 11 reaches the spectrometer 9 through an excitation filter 10 . Light is irradiated to the array 7 through the lens 8 . The fluorescence emitted from the array 7 is collected in the spectroscope 9 through the lens 8 and then reaches the CCD camera 13 through an emission filter 12 . The camera is connected to the CPU 14 to form an image on the VCR 15 and the monitor 16 to analyze specific materials. 6 is a measuring method based on ellipsometry, and an ellipsometry is a method of measuring a change in polarization with respect to a wavelength of reflected light. The combination of the capture probes and the specific material in the spot increases the density of the spot site and changes the polarization of the plane-polarized light beam reflected from the surface, which is monitored directly in solution. It is possible. A typical method is that the monochromatic light emitted from a He-Ne laser ( 17 ) is converted to surface polarized by a polarizer ( 18 ) and irradiated onto the surface of the sample, and then the detector (detector) ( 21 ) is reached. Compensator 19 converts the elliptically polarized reflected beam into surface polarization. Corresponding angles are measured by an analyzer ( 29 ) to calculate the ellipsometric parameters, Psi and Delta, from which the specific material bound to the capture probe is identified. (Azzam et al. , Ellipsometry and Polarized Light, North-Holland Publishing Company: Amsterdam, 1977).

In addition, the present invention is a single-stranded nucleic acid array made of a capture probe containing a specific binding sequence for E. coli, an assay reagent containing a single-stranded nucleic acid or 0.2% BSA, which is used in the reaction of the target probe and E. coli, It provides an analysis kit for the identification and quantitative analysis of E. coli, including hybridization solution, 5'-primer, Cy-5 attached 3'-primer used in the coupling reaction between the target probe and the capture probe.

The present invention provides a system for searching and analyzing biomolecules including microorganisms, cells, and proteins in a sample using a single-stranded nucleic acid array.

The system for searching and analyzing the biomolecules and the like is composed of a method for detecting a single-stranded nucleic acid fixed array, analytical reagent, and a labeling substance of the present invention. The biomolecules include microorganisms such as bacteria and viruses, cell lines, proteins or enzymes isolated therefrom, and the like.

The single-stranded nucleic acid-fixed array means an array in which a single-stranded nucleic acid (capture probe) of the present invention composed of a reactor-spacer site-target probe recognition site is fixed on a slide glass, and the related reagent is the selection buffer, 0.2 It includes a selection buffer containing% bovine serum albumin (BSA) or an assay reagent containing a single nucleic acid strand, and the method of searching for a signal material may use a laser-induced fluorescence method or an elliptical polarization detection method. Bioinformatics can be analyzed and used, and a preferred embodiment of the present invention means laser-induced fluorescence, and biomolecules can be analyzed using the system for identification and analysis.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1. Selection of single stranded nucleic acid and construction of single stranded nucleic acid array

Selection of single-stranded nucleic acid was carried out in the same manner as described in the patent application No. 10-2002-0064027 filed by the inventors, the single-stranded nucleic acids of Table 1 and Table 2 were isolated.

division Nucleotide sequence of N60 of single stranded nucleic acid ( ata cca gct tat tca att (N60) aga tag taa gtg caa tct ) E1 CTT TCT TAC TCG GAT GAT CCT CTT GTG GTC GTC ATA GTC GTT GAA TAC GCT ATT GGG TTG E2 GGT GTG GCA AGG GGT AAC GGT GCG GGA ACG CTA TTC TCG TGT GAT GGT GTA GCC TTG GAA E3 AGT TCG ATG AGG GTG ACA CCG CCA GGA GTG TTT GCT AGA CTT GAG GAA AGG GGC CCG GTG E4 ACC CGT CGA TAA TGA CTG AAC TTC CTC TAT CTT AAA GGG GGT GCG CTT GTT GGG AAG GAG E5 GGG TAA GGG GAT GTT TCT GGG ATT CAA GCG CCT GAT TCT GAG GTG GGC CTC CGC TAC GAG E6 TCT GTA TTT GTA CGC ACC GAA GAT AAG AGA GGG AGT GGA TGG GCT GAA ATG GAG GAC AGG E7 CAT GGG CGG GCC GCG GTC TAT TCG GGA TTA TTG CGG ATC CAA GCG TTT TTG GTG TAT GAG E8 TCA GGG TGT GAA GTT CTT CCG CGC TAG TGG CTT GTA TGT TAT GGA TAA TAC CGC CTC ATG E9 GTC GGG GTT GCA AGG CGT CGT AGC GTG TAT TTG TGA TGG TGA GGA GGT GAA AGG CTG TGA E10 GCG TAT GGG CGG GTC ATT AGG ACA ATT TAA CCA CTC GCG AAA GGG GGC AGC TTG AGT TGG E11 ATT GTC TGT GTG ACT AGT CGG TCT AGT GTG GGG GAG AAG AGT AAA CAA TCG GCG GTG AAG E12 TTA CCA CTG AGT TAA TTT GTA CGG TCT GCG GTG TAC TTT AAG TAT GAA GGG GAC TAC ACA E13 GCT ATC AAT ATT ATA GAG GCG GTC GGG GTA GTG TCA TCG TGG ATA TTT TTT ATG GGC GGG E14 TAG GAG AGC GGG AGC TGA GAA CTT AGA GGC GCC GAT ACA CGA GGA CGG ATT TCT CAA TGG E15 GAC GTA TTA CAG TTA AGT TGG CGC CAT TCG ATT TCT GAT CAC CGG CCG GCA TCA GAG GGG E16 ATA CCA GCT TAT TCA ATT ATA CCA GCT TAT TCA ATT TTG TGG GTG GAT TGG TTG GTG TAG E17 CCG TAA GTC CGG TCT TCC TTG CTG AGT CGC CCT TTC AGG TGC CCA AGC TAC GTG CTG AGA E18 TTG GTG GGG AGG GCC AGT TAG GTC TAA TTT CCG ACG CGC AAG AAT GTC TAC TCA GGT CCG

division Nucleotide sequence of N60 of single stranded nucleic acid ( ata cca gct tat tca att (N60) aga tag taa gtg caa tct ) N1 GTC TAC AAA GGC GAG ACA TGT GCT GTA AAC GTT TTT GTA ATA AGA GTC CGC GTT AAT CCG N2 CTG CTA CGC GAG GCG GGT AAA TTC GGT GGG CTA GAG GCG AGC GAC ATA GTA GTG TAG GGA N3 TAT TTG TAG GGG GCT ACT GGT TAA CGT AAT TGG TCT TTA AGG AGG TTT AGT CGG TGG GTG N4 TAT AAA GGC GGG GTG CAT AGT TGG GAC GGG AGA ATC TTT GGG GCG ATG TTT TTG GGT GCG N5 TCC GGG AGA CGG GTG GTG CGT GTG CCT TTC GAT AGT TAA CGG GGA TGA CCA AAT GCA TAG N6 TCG CAG TTG TGG CTA ATG GGT GAG AGG GAG TGA GTG TTC TAT AAA TAA TTC GAA AAG TCC

Example 2. Preparation of single stranded nucleic acid array

      The single stranded nucleic acid (capture probe) to be immobilized on the surface of the glass slide is a template of the cloned plasmid of the selected single stranded nucleic acid as a template, and the 5'-primer and the 3 'side modified with an amine group. Prepared by performing standard PCR with primers. Isolated 1 pg plasmid, PCR reaction solution, 100 pM 5'-primer, 100 pM 3'-primer, dNTP mixture (5 mM dATP, 5 mM CTP, 5 mM dGTP, 5 mM dTTP) at 94 ° C for 30 seconds, Double-stranded nucleic acid was synthesized by repeating 30 cycles at 52 ° C. 30 seconds and 72 ° C. 20 seconds. In accordance with the protocol of the PCR Product Purification Kit (QIAquick PCR Purification Kit, Qiagen), unbound primers were removed and a capture probe was prepared to be immobilized on the slide. Glass slides were prepared using an aldehyde-plated slide, CSS (BMS), as an array in which single-stranded nucleic acids were arranged as shown in FIG. 7. The array was fabricated using a pin-type microarrayer system (GenPak, Inc.), and the spot spacing was such that the center-to-center was 370 μm. Each single stranded nucleic acid was dissolved in 3xSSC buffer to adjust the concentration. At this time, the humidity in the arrayer was maintained at 70% to perform spotting. The spotted slides were left in a humidified chamber for 15 minutes and then baked at 80 ° C. for 2 to 4 hours to perform baking. After the single-stranded nucleic acid was fixed to the glass slide by a known method, the slide was immediately centrifuged and dried, and then stored in a light-blocked state.

Example 3. Preparation of specific substance and target probe complex

      Single-stranded nucleic acids used as target probes were composed of plasmids cloned with single-stranded nucleic acids E1 to E18 and N1 to N6 selected as SELEX, and the fluorescent label Cy5 (sulfoindocyanine) attached to the 5 'side. Prepared by asymmetric PCR using '-primer (SEQ ID NO: 1) and 3'-primer (SEQ ID NO: 2). Standard asymmetric PCR method in isolated 1 pg plasmid, PCR reaction solution [5'-primer with 10 pM Cy5, 1 pM 3'-primer, dNTP mixture (5 mM dATP, 5 mM CTP, 5 mM dGTP, 5 mM dTTP)] 30 cycles were repeated at 94 ° C. 30 sec, 52 ° C. 30 sec, and 72 ° C. 20 sec to fluoresce the single stranded nucleic acid. Unbound bases and primers were removed according to the protocol of the PCR Product Purification Kit (QIAquick PCR Purification Kit, Qiagen).

Specimen samples were prepared by diluting Escherichia coli and colony forming units (cfu) were measured according to standard methods.

Specifically, after mixing the purified target probe solution and the 150μL selection buffer (0.2% BSA) containing the sample and reacted for 30 minutes at 37 ℃, E. coli-target probe complex by centrifugation of the selection buffer or 50 mM Washed three times with EDTA and the reaction product was harvested by centrifugation for 15,000xg, 5 minutes. The hybridization solution was added thereto, heated at 95 ° C. for 5 minutes, and then treated with ice.

Example 4 Isolation of the Complex and Reaction of the Capture Probe with the Target Probe

The hybridization solution was treated on the glass slide with single-stranded nucleic acid prepared in Example 2 and reacted at 42 ° C. for 30 minutes. The hybridization solution then comprises 1 M sodium chloride, 0.3 M sodium citrate, 0.5% SDS or 100 μg / ml salmon pump DNA, 0.2% bovine serum albumin or single stranded nucleic acid. The hybridized glass slides were treated with the solution prepared in Example 3, hybridized at 42 ° C. for 12 hours, and then the array was washed with a washing solution. At this time, the composition of the washing solution is, for example, 1X SSC + 0.2% SDS, 1X SSC + 0.2% SDS, 0.5X SSC + 0.2% SDS, 0.01X SSC + 0.2% SDS in the order of 30 ℃ at each step 30 It was performed for a minute.

Example 5 Exploration and Analysis

After the washing of Example 4 was completed, the glass slide was dried by centrifugation, and then a laser scanner having a laser (Cy5, 635 nm) of a suitable wavelength for excitation of the used fluorescent dye (laser scanner, GenePix4000, Search using Axon). The fluorescence images were saved in a multi-image-tagged image file (TIFF) format and analyzed with an appropriate image analysis software (GenePix Pro 3.0, Axon) (FIG. 3). ). The signal intensity per spot (quanta) is used by subtracting the base signal of the surrounding background, where the background signal is a local background consisting of the surrounding signals of four spots around a particular spot. ). If more than 90% of the pixels in the spot show a signal intensity that exceeds the background signal + 2 standard deviation (SD), then it is included in the data analysis. It is common to classify as a bad spot and not include it in the analysis. Variation according to labeling efficiency is normalized (eg, Normalized Intensity = Probe Intensity / IS intensity) using the signal of the internal standard (IS), and in the case of monolabeling The result is recorded as the signal intensity of the Cy5 channel and the average value is used when the spotting is more than doubled. The signal intensity (S) of the probe is obtained by obtaining the respective signal intensities for the pixels of the spot and then using their median value (median value of pixel-by-pixel). Signal intensity (S) is averaged to the deviation according to labeling efficiency using the internal standard (IS).

S '(normalized value) = S (Cy5-reference) x (Cy5-IS).

The above method can provide a method for quantifying the number of E. coli in the sample from the array fluorescence data by identifying the relationship between the result of analyzing the pixel density and the actual sample amount, and forming the correlation. Can be analyzed by clustering method.

The degree of fluorescence varies depending on the spot caused by the double strands formed by the capture probes and the target probe. The specific substance-target probe complex has a degree of binding and an amount of binding depending on the specificity and binding strength therebetween. The sequence of target probes and capture probes dissociated from the specific material-target probe complex affects the stability of the target probe-capture probe double strand, and the amount of target probe affects the degree of fluorescence. Therefore, by analyzing the degree of fluorescence of the formed spot it is possible to determine the type and quantitative change of the specific material.

The results of the above test are shown in FIGS. 8A and 8B, and as shown in FIG. 8A and FIG. Controls (N1-6, SEQ ID NOs: 21-26) did not have fluorescence. In addition, when tested with the same method using Listeria monocytogenes ( ATCC # 19118) as a sample, the fluorescence could not be confirmed. From the above results, it was confirmed that the nucleotide sequence of E1 to 18 (SEQ ID NOs: 3 to 20) selected as SELEX has a specific binding force to E. coli.

In addition, as compared with the case of E. coli 100 cfu (Fig. 8a) and the case of E. coli 10,000 cfu (Fig. 8b), the fluorescence is relatively higher than the case of E. coli 10,000 cfu 100 cfu than the difference in fluorescence cfu It can be confirmed that there is a correlation. That is, double strands of target probes are formed on the capture probe, and the degree of fluorescence is different according to the number of double strands, which is correlated with the number of microorganisms.

By the above method, the single-stranded nucleic acid attached array having recognition sites for various materials can be used to search for and analyze the identification and quantitative changes of various materials in one sample.

In the present invention described above, the single-stranded nucleic acid, which is an aptamer, forms a complex with a specific substance according to a given environment, or identifies and quantitatively changes a specific substance using a dual function of forming complementary single-stranded and double-stranded nucleic acids. Analytical methods can be used to find and analyze microorganisms, cells, proteins containing biomolecules, etc. Ultimately, they can be widely applied to medicine, veterinary medicine, environmental engineering, food engineering, and agriculture.

1 is a diagram illustrating the dual function of aptamer single-stranded nucleic acid forming a complex with a specific substance in a Selex selection buffer or forming a single-stranded and double-stranded nucleic acid complementary to a nucleic acid hybridization solution.

2 is a diagram of an array constructed using single-stranded nucleic acid,

3 is a diagram of the shape of a single stranded nucleic acid attached to an array,

4 a is a diagram illustrating a procedure of analyzing a sample by the aptamer probe method,

4 b is a diagram illustrating a procedure of analyzing a sample by the anti-aptamer probe method,

5 is a diagram of a laser fluorescence triggering system for analyzing the spots of an array;

FIG. 6 is a diagram of an ellipsometric detection system for analyzing the spots of an array; FIG.

7 is a diagram of an array of single stranded nucleic acids that are capture probes immobilized on a single stranded nucleic acid array,

Figure 8a is a result of E. coli 100 cfu analysis with a single stranded nucleic acid array and a single stranded nucleic acid,

Figure 8b is a result of E. coli 10,000 cfu analysis with a single stranded nucleic acid array and a single stranded nucleic acid.

<110> GENOPROT CO., LTD. KIM, Sung Chun <120> Method for identification and analysis of certain molecules using the dual function of single strand nucleic acid <160> 26 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 5'-primer <400> 1 ataccagctt attcaatt 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 3'-primer <400> 2 agattgcact tactatct 18 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> E1: nucleic acid lignd to E. coli <400> 3 ctttcttact cggatgatcc tcttgtggtc gtcatagtcg ttgaatacgc tattgggttg 60 60 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 4 ggtgtggcaa ggggtaacgg tgcgggaacg ctattctcgt gtgatggtgt agccttggaa 60 60 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 5 agttcgatga gggtgacacc gccaggagtg tttgctagac ttgaggaaag gggcccggtg 60 60 <210> 6 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 6 acccgtcgat aatgactgaa cttcctctat cttaaagggg gtgcgcttgt tgggaaggag 60 60 <210> 7 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 7 gggtaagggg atgtttctgg gattcaagcg cctgattctg aggtgggcct ccgctacgag 60 60 <210> 8 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 8 tctgtatttg tacgcaccga agataagaga gggagtggat gggctgaaat ggaggacagg 60 60 <210> 9 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 9 catgggcggg ccgcggtcta ttcgggatta ttgcggatcc aagcgttttt ggtgtatgag 60 60 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 10 tcagggtgtg aagttcttcc gcgctagtgg cttgtatgtt atggataata ccgcctcatg 60 60 <210> 11 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 11 gtcggggttg caaggcgtcg tagcgtgtat ttgtgatggt gaggaggtga aaggctgtga 60 60 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 12 gcgtatgggc gggtcattag gacaatttaa ccactcgcga aagggggcag cttgagttgg 60 60 <210> 13 <211> 60 <212> DNA <213> Artificial Sequence <220> E11: nucleic acid ligand to E. coli <400> 13 attgtctgtg tgactagtcg gtctagtgtg ggggagaaga gtaaacaatc ggcggtgaag 60 60 <210> 14 <211> 60 <212> DNA <213> Artificial Sequence <220> E12: nucleic acid ligand to E. coli <400> 14 ttaccactga gttaatttgt acggtctgcg gtgtacttta agtatgaagg ggactacaca 60 60 <210> 15 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 15 gctatcaata ttatagaggc ggtcggggta gtgtcatcgt ggatattttt tatgggcggg 60 60 <210> 16 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 16 taggagagcg ggagctgaga acttagaggc gccgatacac gaggacggat ttctcaatgg 60 60 <210> 17 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 17 gacgtattac agttaagttg gcgccattcg atttctgatc accggccggc atcagagggg 60 60 <210> 18 <211> 60 <212> DNA <213> Artificial Sequence <220> E16: nucleic acid ligand to E. coli <400> 18 ataccagctt attcaattat accagcttat tcaattttgt gggtggattg gttggtgtag 60 60 <210> 19 <211> 60 <212> DNA <213> Artificial Sequence <220> E223: nucleic acid ligand to E. coli <400> 19 ccgtaagtcc ggtcttcctt gctgagtcgc cctttcaggt gcccaagcta cgtgctgaga 60 60 <210> 20 <211> 60 <212> DNA <213> Artificial Sequence <220> E18: nucleic acid ligand to E. coli <400> 20 ttggtgggga gggccagtta ggtctaattt ccgacgcgca agaatgtcta ctcaggtccg 60 60 <210> 21 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> N1 <400> 21 gtctacaaag gcgagacatg tgctgtaaac gtttttgtaa taagagtccg cgttaatccg 60 60 <210> 22 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> N2 <400> 22 ctgctacgcg aggcgggtaa attcggtggg ctagaggcga gcgacatagt agtgtaggga 60 60 <210> 23 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> N3 <400> 23 tatttgtagg gggctactgg ttaacgtaat tggtctttaa ggaggtttag tcggtgggtg 60 60 <210> 24 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> N4 <400> 24 tataaaggcg gggtgcatag ttgggacggg agaatctttg gggcgatgtt tttgggtgcg 60 60 <210> 25 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> N5 <400> 25 tccgggagac gggtggtgcg tgtgcctttc gatagttaac ggggatgacc aaatgcatag 60 60 <210> 26 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> N6 <400> 26 tcgcagttgt ggctaatggg tgagagggag tgagtgttct ataaataatt cgaaaagtcc 60 60

Claims (8)

(Iii) attaching capture probes, which are single-stranded nucleic acids consisting of a reactor (X) -spacer site (R) -target probe recognition site (V), onto a solid substrate to fabricate an array; (Ii) a second step of preparing the specific substance-target probe complex by reacting the specific substance with the target probe by treatment of the assay reagent; (Iii) a third step of washing and separating the specific material-target probe complex prepared in the second step; (Iv) adding a hybridization solution to the separated complex and treating it for 5 minutes at 95 ° C. to dissociate the target probe; (Iii) a fifth step of reacting the capture probe with the labeled target probe; And (iii) a sixth step of searching for a substance labeled on the target probe, for identifying and quantifying a specific substance. The method of claim 1, wherein the assay reagent of the second step (Iii) 20 to 100 mM tris hydrochloric acid (pH 7.4), (Ii) 0 to 200 mM potassium chloride, sodium chloride, magnesium chloride, or a mixed solution thereof, respectively; (Iii) 0.05 to 0.2% sodium azide, (Iii) a 0.1% to 0.3% bovine serum albumin or a Selectex buffer comprising a nucleic acid that inhibits nonspecific binding. The method of claim 2, wherein the assay reagent is 50mM tris hydrochloric acid (pH 7.4), 5mM potassium chloride, 100mM sodium chloride or 1mM magnesium chloride, 0.1% sodium azide, 0.2% bovine serum albumin or single-stranded nucleic acid . The method of claim 1, wherein the specific substance of the second step is a group consisting of bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies and enzymes isolated therefrom. At least one member selected from. The method of claim 1, wherein the washing buffer of the third step is a 0 to 1 x Serex buffer or 0 to 500 mM EDTA solution. According to claim 1, wherein the nucleic acid hybridization solution of the fourth step (Iii) 0.5 to 1.5 M sodium chloride, (Ii) 0-1.0 M sodium citrate, (Iii) 0.1-0.3% bovine serum albumin, 0-1.0% SDS, 0-10 times Denhart solution or 0-1% nonfat dried milk, a substance that inhibits nonspecific binding milk), (Iii) a nucleic acid that inhibits nonspecific binding, (v) 30 to 60% (v / v) formamide. The method of claim 6, wherein the nucleic acid hybridization solution is 1 M sodium chloride, 0.3 M sodium citrate, 0.5% SDS or 100 μg / ml Salmon's perm DNA, 0.2% bovine serum albumin or single stranded nucleic acid. Single-stranded nucleic acid arrays made of capture probes containing specific binding sequences for E. coli, single-stranded nucleic acids used for the binding reaction between target probes and E. coli, or reagents containing 0.2% BSA, target probes and capture probes. Analysis kit for identification and quantitative analysis of E. coli, including hybridization solution, 5'-primer, Cy-5 attached 3'-primer used in the coupling reaction.