KR20040035248A - Method for identification and analysis of certain molecules using single strand nucleic acid array and nucleic acid ligands to e. coli therefor - Google Patents

Abstract

PURPOSE: A method for identification and analysis of certain molecules using single strand nucleic acid array and nucleic acid ligands to E. coli therefor are provided, thereby rapidly identifying and analyzing the certain molecules from various samples. CONSTITUTION: A method for identification and analysis of certain molecules comprises the steps of: (i) fixing capture probes as single strand nucleic acids consisting of reaction group(X)-spacer domain(R)-recognition site(V) on a solid substrate to prepare an array; (ii) reacting a reaction product between reagent treated certain molecules and target probes with the single strand nucleic acid treated array; and (iii) screening the target probe-labeled molecules, wherein the certain molecule is bacteria, fungus, virus, cell line, tissue, protein isolated therefrom, carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody and enzyme; the capture probe is 5'-primer having the nucleotide sequence set forth in SEQ ID NO:1 wherein 5' is modified by amine, single strand nucleic acid nucleotide sequence having the nucleotide sequences set forth in SEQ ID NO:3 to SEQ ID NO:26 and 3'-primer having the nucleotide sequence set forth in SEQ ID NO: 2; and the reagent consists of 50mM tris HCl(pH 7.4), 5mM potassium chloride, 100mM sodium chloride, 1mM magnesium chloride, and 0.1% sodium azide and is buffer solution containing 0.2% bovine serum albumin or single strand nucleic acid.

Description

Method for identification and analysis of certain molecules using single strand nucleic acid array and Nucleic acid ligands to E. coli therefor}

The present invention relates to a method for identifying and quantitative analysis of a specific substance, and more specifically, to search and analyze for identification and quantitative change of a specific substance using capture probes and target probes, which are single-stranded nucleic acids attached to an array. It's about a simple new way.

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.

Escherichia coli is a bacterium that resides in the intestines of warm-blooded animals and has a Gram-negative flagella. It is not usually pathogenic in the intestine, but enters the urethra or biliary tract, causing cystitis, pyelitis, and cholecystitis. In children, it causes acute enteritis. It is an indicator of fecal contamination and is often used for water quality tests.

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 with a very high affinity not only to proteins that can bind nucleic acids in nature, but also to many biomolecules, including proteins that do not bind nucleic acids. 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.

Therefore, the inventors attached single-stranded nucleic acid to a glass slide and performed reaction and washing using labeled target probes, and then identified specific substances and quantitative changes by laser-induced fluorescence. Was completed.

An object of the present invention is to identify and quantitatively change specific substances by using selected single-stranded nucleic acid-attached glass slides and fluorescently labeled target probes, thereby using a variety of living organisms including microorganisms, cells, and proteins. It is an attempt to provide a system for searching and analyzing molecules.

1 is a diagram of an array fabricated using a single stranded nucleic acid,

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

3 is a diagram showing a method of analyzing a sample using a single stranded nucleic acid array and target probes,

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

5 is a diagram of an ellipsometric detection system for analyzing the spots of an array;

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

7a is a result obtained by reacting a complex of a single stranded nucleic acid array with E. coli 100 cfu and a target probe,

FIG. 7B is a result obtained by treating a single stranded nucleic acid array by treating a complex of E. coli 1000 cfu and a target probe.

In order to achieve the above object, the present invention comprises the steps of: (i) preparing an array by fixing a single stranded nucleic acid consisting of a reactor (X) -spacer site (R) -recognition site (V) on a solid substrate; (Ii) a reaction between the reactant between the specific material and the target probe and the single stranded nucleic acid array; (Iii) Provides a method for identifying and quantitatively identifying a specific substance using a single stranded nucleic acid array, comprising searching for a substance labeled on a target probe.

The specific substance includes bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, enzymes, and the like.

The present invention also provides oligonucleotides set forth in SEQ ID NOs: 3 to 20, which have a specific binding capacity to E. coli.

The present invention provides an analytical reagent for specific binding between a single-stranded nucleic acid capture probe, a target probe, and a specific substance in identification and quantitative analysis of a biomolecule using an array to which a single-stranded nucleic acid is attached.

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 E. coli identification and quantitative analysis, including 5'-primer, 3'-primer with Cy-5 attached.

The present invention also provides a system for searching and analyzing biomolecules, including a method for searching for analyte reagents and labeling agents such as single-stranded nucleic acid arrays, selection buffers, and the like.

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

Identification and quantitative analysis of a specific substance according to the present invention consists of a single-stranded nucleic acid array manufacturing step (iii), reaction step (ii), search step (iii).

The first step of the present invention is an array fabrication step (ⅰ) attached to a single stranded nucleic acid is as follows.

The present invention provides a single stranded nucleic acid consisting of three parts, a capture probe to be fixed to a single stranded nucleic acid array, a reactor (X) -spacer site (R) -recognition site (V).

The recognition site is a nucleotide sequence of a single-stranded nucleic acid selected by SELEX, a site for recognizing a specific substance, a single-stranded nucleic acid having a nucleotide sequence of 50 to 100bp, preferably in the present invention SELEX standard method (Bock LC et al .; Nature , 355 (6360) , pp564-6, 1992). A sequence having a nucleotide sequence isolated from the single-stranded nucleic acid aptamers that specifically binds to E. coli. Base numbers of the numbers 3 to 26;

The present invention also provides oligonucleotides set forth in SEQ ID NOs: 3 to 20, which have a specific binding capacity to E. coli.

In addition, the spacer (spacer) is a site in consideration of the problems 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 5 to 20 amino acid sequences, In a preferred embodiment of the present invention provides a nucleotide sequence of SEQ ID NO: 1.

In addition, the reactor portion is a site to which the reactor is attached to attach the single-stranded nucleic acid to the substrate of the array, the reactor may use an amine (amine) group, thiol (-SH) group, biotin (biotin) and the like.

Preferred single-stranded nucleic acid capture probe of the present invention is a 5'-primer (SEQ ID NO: 1), wherein the 5 'of the nucleic acid is modified with an amine group, the single-stranded nucleic acid sequence of SEQ ID NO: 3 to 26 and the 3'-primer (SEQ ID NO: 2) It provides a single-stranded nucleic acid consisting of a capture probe, which isolates the single-stranded nucleic acid that specifically binds to E. coli among the single-stranded nucleic acids selected by SELEX standard method, and then adjust the primer ratio of SEQ ID NO: 1 and SEQ ID NO: 2 Can be prepared by repeating the asymmetric PCR.

The present invention also provides an analysis method using a single stranded nucleic acid array prepared using capture probes comprising the nucleotide sequences set forth in SEQ ID NOs: 3 to 26.

The substrate may be made of an inorganic material such as glass or silicon, or made of a polymer material such as acrylic or PET (polyethylene terephtalate), polycarbonate, polystyrene, polypropylene, and the like. It means the produced slide glass. The substrate is coated with aldehyde or poly L-lysine or the like.

More specifically, the capture probe to be immobilized on the glass slide surface is a template of a single stranded nucleic acid cloned plasmid selected by the SELEX standard method, 5'-primer and 3 'modified with an amine group on the 5' side. Prepare by performing asymmetric PCR with '-primer. Single stranded nucleic acid by standard asymmetric PCR on isolated 1 pg plasmid, PCR reaction solution, 10 pM 5'-primer, 1 pM 3'-primer, dNTP mixture (5 mM dATP, 5 mM CTP, 5 mM dGTP, 5 mM dTTP) Synthesize Unbound primers are removed with a PCR product purification kit, and single stranded nucleic acid is prepared, which is a capture probe to be immobilized on a slide. 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 × SSC buffer to adjust the concentration, and spotting is performed by keeping the humidity in the arrayer at 70 to 80%. 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.

1 is an arrangement of single stranded nucleic acids attached to the array, with spot 1 at the surface of the array. FIG. 2 is a spot-shaped view attached to an array in which single-stranded nucleic acid 5 is attached to monolayer 3 coated on substrate surface 2 and single-stranded nucleic acid 5 is attached to monolayer 3 coated with spacer 4 .

The present invention provides a method for producing a capture probe that is a single stranded nucleic acid.

The present invention also provides a method for producing a single stranded nucleic acid array.

The reaction step (ii) of the analytical method of the present invention is composed of a first reaction, a second reaction, and a third reaction, and it is ideal to perform the division, except that the second reaction may be performed or may be performed at the same time. Is the same as FIG.

In the reaction step (ii), the target probe is prepared by labeling with a single-stranded nucleic acid which binds to a specific substance, and various kinds of labeling substances capable of smoothly recognizing a signal may be used in the searching step. The target probe is a single-stranded nucleic acid selected through SELEX or a single-stranded nucleic acid having a structure capable of specific binding with a target substance. For this purpose, a secondary structure and structure free energy using an MFOLD program that models a secondary structure of nucleic acid. After confirming, single-stranded nucleic acid having the highest stability secondary structure is selected.

As a label of the target probe, a fluorescent substance such as radioisotope, fluorescein, Cy3 or Cy5, or biotin may be used, and preferably, a fluorescent substance is used. The specific substance includes bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, enzymes, and the like isolated therefrom. . The cell lines and tissues include those derived from prokaryotes or eukaryotes, and eukaryotes refer to humans, animals and plants.

The first reaction is a reaction between a specific substance and a target probe, which is a single stranded nucleic acid, and the reaction solution is composed of a salt composition capable of binding the single stranded nucleic acid to a specific substance well, and an element that prevents nonspecific binding. Preferably a selection buffer containing 50 mM Tris hydrochloric acid (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1% sodium azide and 0.2% bovine serum albumin or mononucleic acid strands added thereto. Use an assay reagent containing a. 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 30 ℃. In general, the protein and single-stranded nucleic acid is excessively treated to prevent nonspecific binding of the target probe. Preferably, the antisense strand of the target probe is a single-stranded nucleic acid, a yeast tRNA, and a salmon sperm DNA. Alternatively, human placental DNA may be used. Then, the complex of specific substances and target probes and the unbound target probes can be separated by membrane filtration or centrifugation. If the sample is a microorganism or a cell in the first reaction, the target probe solution is mixed with a selection buffer containing the sample and reacted at room temperature for 30 minutes, and then the non-binding target probe is directly removed by centrifugation or filtration using 0.42 μm membrane. It can also be removed and used for the tertiary reaction. In the case of proteins, single-stranded nucleic acids, protein complexes and unbound single-stranded nucleic acids may be separated by a filtration method using a 0.45 μm nitrocellulose membrane filter.

The secondary reaction is a step of reacting and washing excess single-stranded nucleic acid in an array to which a capture probe, which is a single-stranded nucleic acid, is attached. Preferably, the yeast tRNA and salmon sperm DNA are treated with the single-stranded nucleic acid. DNA) or human placental DNA.

The third reaction is a step of washing and drying the first reaction product and the second reaction product.

In addition, in the identification and quantitative analysis of biomolecules using the single-stranded nucleic acid-attached array of the present invention, an assay reagent for specific binding between a single-stranded nucleic acid capture probe and a target probe and a specific substance is provided.

The assay reagent comprises (i) potassium chloride as a substance involved in the secondary structure stabilization of tris hydrochloric acid (pH 7.4), (ii) single stranded nucleic acid at 20 to 100 mM, preferably 50 mM for buffering pH. , Sodium chloride and magnesium chloride, respectively, 0 to 200 mM, preferably 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, and (iii) 0.05 to 0.2% sodium azide for bacterial propagation inhibition. Preferably 0.1% sodium azide and (iii) single stranded nucleic acid or nonspecific having a molar number of 1 to 10-fold based on 0.1% to 0.3%, preferably 0.2% bovine serum albumin or target probe for nonspecific binding inhibition A nucleic acid that inhibits phosphorus binding 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.

In a preferred embodiment of the present invention, a reagent containing 50 mM tris-hydrochloric acid (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1% sodium azide or single stranded nucleic acid is used as an assay reagent.

The capture probe, the specific substance and the target probe complex are determined by the specificity and the binding force between them, from which specific substances can be identified and quantified. The specificity and binding strength of the probe and the specific substance change the degree of binding of the complex, which affects the detection result. Therefore, by analyzing the detection degree, it is possible to confirm the kind and quantitative change of a specific substance.

The search step (iii), which is the third step of the analytical method of the present invention, is to perform identification and quantitative analysis of a specific substance, and various methods such as FIG. 4 and FIG. You can choose.

4 shows that after placing the array 7 on the plate 6 , the light irradiated from the lamp 11 reaches the spectroscope 9 through an excitation filter 10 . Light is emitted to array 7 through lens 8 . The fluorescence from the array 7 is collected in the spectroscope 9 through the lens 8 and then reaches the CCD camera 13 through the emission filter 12 . The camera is connected to the CPU 14 and can be imaged on the VCR 15 and the monitor 16 to analyze specific materials.

5 is a measurement method based on ellipsometry, and the 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 monochromatic light irradiated from a He-Ne laser 17 is converted to plane polarized by a polarizer 18 and irradiated to the surface of the sample, and then detector 21 Is reached. Compensator 19 converts the elliptically polarized reflected beam into surface polarization. Corresponding angles are measured by analyzer 29 to calculate the ellipsometric parameters Psi and Delta, from which the specific material bound to the capture probe can be identified. (Azzam et al. , Ellipsometry and Polarized Light, North-Holland Publishing Company: Amsterdam, 1977).

This method is useful as a system for searching and analyzing biomolecules including microorganisms, cells and proteins.

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 used in the binding reaction of the array, target probe and E. coli Provides an analysis kit for E. coli identification and quantitative analysis, including 5'-primer, 3'-primer attached to Cy-5.

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 consisting of a reactor-spacer site-recognition site is fixed on a slide glass, and the related reagent is 0.2% of the selection buffer. An analysis reagent containing a selection buffer or a single nucleic acid strand containing serum albumin (BSA), and a method for searching for a signal material may use laser-induced fluorescence or elliptical polarization detection, and a preferred embodiment of the present invention. By laser induced fluorescence method, it is possible to analyze the biomolecules using the system for identifying and analyzing.

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

SELEX libraries, primers and single stranded nucleic acids were prepared as follows. The HPLC purified SELEX library contains a nucleotide sequence of 60 nucleotides randomly arranged between a primer binding sequence of 18 nucleotides (5'-ATA CCA GCT TAT TCA ATT (N) 60 AGA TAG TAA GTG CAA TCT-3 ': MWG). -Biotech AG). 5'-primer (SEQ ID NO: 1) (5'-ATA CCA GCT TAT TCA ATT-3 '; spacer) and 3'-primer (SEQ ID NO: 2) (5'-AGA TTG CAC TTA CTA TCT-3' ) Was used as a reaction primer when PCR (polymerase chain reaction) for the synthesis of double-stranded or single-stranded DNA. Single-stranded nucleic acids were prepared by performing asymmetric PCR ((Asymmetric PCR) by adjusting the ratio of the 5'- primer and 3'-primer.

Selection of aptamers, single-stranded nucleic acids for the target material, was performed by SELEX standard method (Bock LC et al .; Nature , 355 (6360) , pp564-6, 1992). Aptamers are oligonucleotides that show specific binding ability to a specific substance, and thus, a highly specific sequence can be obtained by repeating a selection process.

The single-stranded nucleic acid synthesized in the above was selected at a concentration of 10 ¹⁵ nucleotide sequences / 1 ml, and at a concentration of 200 pmol / 200 μl from the next time (50 mM TrisCl (pH 7.4), 5 mM KCl, 100). mM NaCl, 1 mM MgCl ₂ , 0.1% NaN ₃ ) was heated at 80 ° C. for 10 minutes, and then left on ice for 10 minutes. The reaction solution was prepared by adding 5 times yeast tRNA (yeast tRNA, Life Technologies) and 0.2% BSA (bovine serum albumin, Merck) of the used single-stranded nucleic acid. Single-stranded nucleic acid was added in a selection buffer containing 10 ⁶ Escherichia coli DH-5α (Amershambiosciences) and reacted at 37 ° C. for 30 minutes. Single-stranded nucleic acid binding to E. coli was centrifuged and separated, and washed with 1 ml selection buffer (containing 0.2% BSA). Solution I (selection buffer, 10 mM EDTA) was treated with the reaction to separate the single stranded nucleic acid, followed by complete separation of the single stranded nucleic acid by phenol extraction and ethanol precipitation.

In order to perform the second process, the single stranded nucleic acid obtained by ethanol precipitation was subjected to asymmetric PCR (asymmetric PCR) using a 5'-primer and a 3'-primer. It was performed repeatedly. After performing the final eight times, the final isolated single-stranded nucleic acid was PCR using 5'-primer and 3'-primer to synthesize double-stranded nucleic acid. The PCR product was cloned into a T-vector to determine the nucleotide sequence. Then, 18 single-stranded nucleic acids having high binding ability with E. coli were selected, and E1-18 (SEQ ID NOs: 3-20) were selected. Named N1-6 (SEQ ID NOS: 21-26). Table 1 lists the nucleotide sequences E1 to E18 of the single-stranded nucleic acids of the present invention, Table 2 lists the nucleotide sequences N1 to N6 of the single-stranded nucleic acids of the present invention.

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 Strand Nucleic Acid Array

The single stranded nucleic acid (capture probe) to be immobilized on the glass slide surface is a 5'-primer and 3 'modified with an amine group on the 5' side by using a templated clone of the selected single stranded nucleic acid. Prepared by performing asymmetric PCR with primers. Isolated 1 pg plasmid, PCR reaction solution, 10 pM 5'-primer, 1 pM 3'-primer, dNTP mixture (5 mM dATP, 5 mM CTP, 5 mM dGTP, 5 mM dTTP) at 94 ° C for 30 seconds 30 cycles were repeated at 52 ° C., 30 seconds, and 72 ° C. for 20 seconds to synthesize single-stranded nucleic acids. According to the protocol of the PCR product purification kit (QIAquick PCR Purification Kit, Qiagen), unbound primers were removed, and single-stranded nucleic acid, which was a capture probe to be immobilized on a slide, was prepared. Glass slides were prepared using an aldehyde-plated slide, CSS (BMS), to prepare an array of single-stranded nucleic acids as shown in FIG. 6. 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 3 × SSC 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: Reaction

The single-stranded nucleic acid used as target probes was a 5'-primer (SEQ ID NO: 5) attached with a fluorescent label Cy5 (sulfoindocyanine) on the 5 'side using a plasmid cloned with E5, a single-stranded nucleic acid selected from SELEX. 1) and 3'-primer (SEQ ID NO: 2) was used to prepare by asymmetric PCR. 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 under conditions of 94 ° C. 30 sec, 52 ° C. 30 sec, and 72 ° C. 20 sec. Unbound bases and primers were removed according to the protocol of the PCR Product Purification Kit (QIAquick PCR Purification Kit, Qiagen).

The reaction was carried out in three steps: first reaction, second reaction and third reaction. Samples were prepared by diluting E. coli (DH-5α) for 12 hours, diluting them, and measuring colony forming units (cfu) according to standard methods.

In the first reaction, the purified target probe solution and the 150 μl selection buffer containing the sample (containing 0.2% BSA) were mixed and reacted at room temperature for 30 minutes, followed by the single-stranded nucleic acid that is the antisense strand of the target probe. Alternatively, single-stranded nucleic acid (yeast tRNA) was added and reacted at room temperature for 10 minutes.

In the second reaction, the glass slide with the single-stranded nucleic acid prepared in Example 2 was treated with Solution II (selective buffer + single-stranded nucleic acid), reacted for 10 minutes at room temperature, and washed with the selective buffer.

In the third reaction, the first reaction product was treated on the glass slide after the second reaction, and reacted at room temperature for 30 minutes, followed by washing with a selective buffer.

Example 4: Exploration and Analysis

After drying the glass slide after tertiary reaction by centrifugation, a laser scanner (Laser scanner, GenePix4000, Axon) having an appropriate wavelength laser (Cy5, 635 nm) for excitation of the used fluorescent dye was used. The fluorescence images were stored in a multi-image-tagged image file (TIFF) format and analyzed with an appropriate image analysis software (GenePix Pro 3.0, Axon). 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 by the labeling efficiency using the internal standard (IS).

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

By the above method, the relationship between the analysis result of the pixel density and the actual sample amount can be identified and the correlation can be created to provide a method for quantifying the number of bacteria in the sample from the array fluorescence data.

Fluorescence levels vary depending on the complex formed by capture probes, specific materials, and target probes. The capture probe, the specific substance and the target probe complex are determined by the specificity and the binding force between them, from which specific substances can be identified and quantified. The specificity and binding force of the probe and the specific substance change the degree of binding of the complex, which affects the degree of fluorescence. Therefore, by analyzing the degree of fluorescence it is possible to confirm the type and quantitative change of a specific material.

The results of the above test are shown in FIGS. 7A and 7B, and as shown in the aptamer-attached site (E1-18, SEQ ID NOs: 3-20) on E. coli in a glass slide with single-stranded nucleic acid There was fluorescence, but the negative controls (N1-6, SEQ ID NOs: 21-26) were not 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) of FIG. 6 selected as SELEX has specific binding ability to E. coli.

In addition, as a result of comparing 100 cfu of E. coli (Fig. 7a) and 10,000 cfu of E. coli (Fig. 7b), the degree of fluorescence is relatively higher than that of 100 cfu of E. coli 10,000 cfu than the difference in fluorescence of cfu of E. coli It can be confirmed that there is a correlation. That is, the complex of the E. coli and the target probe is attached to the capture probe, and the degree of fluorescence is different according to the number of the complexes, 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.

The present invention described above can be used for the analysis and analysis of biomolecules including microorganisms, cells, and proteins, and the like. Ultimately, it can be widely applied in medicine, veterinary medicine, environmental engineering, food engineering and agriculture.

Claims (9)

(Iii) attaching capture probes, which are single-stranded nucleic acids consisting of a reactor (X) -spacer site (R) -recognition site (V), onto a solid substrate to fabricate an array; (Ii) a reaction between the specific material treated with the assay reagent and the target probe and a reaction with the single stranded nucleic acid array; (Iii) A method of identifying and quantitatively analyzing specific substances using a single stranded nucleic acid array, comprising searching for a substance labeled on a target probe. The method of claim 1, wherein the specific substance is bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, enzymes. The analysis according to claim 1, wherein the capture probe is a probe consisting of a 5'-primer of SEQ ID NO: 1 in which 5 'is modified with an amine group, a mononucleate sequence described in SEQ ID NOs: 3 to 26, and a 3'-primer of SEQ ID NO: 2 Way. The assay reagent of claim 1 has a composition consisting of 50 mM Tris hydrochloric acid (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1% sodium azide, and comprises 0.2% bovine serum albumin or single stranded nucleic acid. Analysis method which is a buffer solution. An oligonucleotide according to SEQ ID NOs: 3 to 20, having specific binding ability to E. coli. In the identification and quantitative analysis of a specific substance using a single-stranded nucleic acid array, an assay reagent containing the following composition for specific binding between a single-stranded nucleic acid capture probe and a target probe and the specific substance; (Iii) 20 to 100 mM tris hydrochloric acid (pH 7.4), (Ii) 0 to 200 mM potassium chloride, sodium chloride or magnesium chloride, and a mixed solution thereof, respectively; (Iii) 0.05 to 0.2% sodium azide, (Iii) 0.1-0.3% bovine serum albumin or single-stranded nucleic acid with a molar number of 1 to 10-fold based on target probe or nucleic acid that inhibits nonspecific binding. The assay reagent according to claim 6, which is 50 mM Tris hydrochloric acid (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1% sodium azide or single stranded nucleic acid. Single-stranded nucleic acid array made with a capture probe containing a specific binding sequence to E. coli, the assay reagent used for the binding reaction between the array and the target probe and E. coli, single-stranded nucleic acid or 0.2% BSA, Cy- Analytical kit for identification and quantitative analysis of E. coli, including 5'-primer, 3'-primer with 5 attached. A system consisting of the following to search for and analyze biomolecules including microorganisms, cells, and proteins in a sample using an array with a single stranded nucleic acid; (Iii) an array in which a single-stranded nucleic acid consisting of a reactor-spacer site-recognition site is fixed to a slide glass, (Ii) the analytical reagent of paragraph 6; (Iii) Laser fluorescence induction detection methods to detect markers.