WO2019050222A1 - Method for simply and efficiently isolating and purifying chemically labeled oligonucleotides by means of ph control - Google Patents

Abstract

The present invention relates to a method for simply and efficiently isolating and purifying chemically labeled oligonucleotides by means of pH control and, more specifically, to a method and a kit for efficiently isolating and purifying labeled nucleotides by using a change in the distribution constant of a chemical marker according to the pH control of a reaction solution.

Description

Simple and Efficient Separation and Purification of Chemically Labeled Oligonucleotides by pH Control

The present invention relates to a simple and efficient separation and purification method of chemically-labeled oligonucleic acid by pH control, and more particularly, to a method for efficiently and efficiently separating and purifying a labeled nucleic acid by using a change in distribution constant of a chemical marker according to pH control of a reaction solution And a kit using the same.

Nucleic acid is crucial to life as a carrier or messenger of genetic information. Since their discovery by F. Miescher, they have been able to elucidate their function, structure and mechanism of action by stimulating a widespread scientific interest.

As knowledge of this basic molecular biological mechanism has increased, a new combination of genes has become available in recent years and this technology opens up new possibilities, for example, in medical diagnosis and treatment, and in plant breeding.

On the other hand, molecular diagnostics is a field of in vitro diagnostics that detects or measures biomarkers such as DNA, RNA, and proteins using molecular biological techniques. In a narrow sense, molecular diagnostics is a DNA or RNA Is used in the same sense as nucleic acid diagnostics for analyzing and detecting nucleic acid. In this field, a labeled nucleic acid in which a fluorescent substance or a biochemical probe is bound has been the subject of interest.

In this connection, a labeled nucleic acid is prepared by reacting a nucleic acid having a nucleophilic functional group such as a primary amine group or a thiol group with a marker such as a fluorescent dye or a probe, which is functionalized with an electrophilic functional group, and analyzing the labeled nucleic acid A variety of methods are known [Hermanson, GT Bioconjugate techniques; Academic Press, 2008.].

For example, in the case of a primary amine-mediated conjugation, a fluorescent dye comprising an amine-reactive functional group capable of reacting with a primary amine group such as ester, isothiocyanate, aldehyde, etc., Chemically react to form a labeled nucleic acid, and the labeled nucleic acid is required to be separated and purified from unreacted dye and unreacted nucleic acid.

Generally, in order to increase the yield of the labeled nucleic acid obtained through the reaction of the marker and the nucleic acid, the marker is used in excess relative to the nucleic acid. Therefore, untagged unreacted nucleic acids are present in trace amounts or almost not exist in the labeling reaction solution of nucleic acid, and unreacted markers are present in excess. A method of separating and purifying these excess unreacted markers from the labeled nucleic acid is indispensable.

In this regard, conventional methods of removing unreacted dyes include ethanol precipitation, size-exclusion chromatography, and dialysis (Giusti, W. G .; Adriano, T. PCR methods and applications 1993, 2, 223] have been known, but these methods generally involve time consuming, inconvenient and inefficient disadvantages.

For example, it takes about 30 minutes to 1 hour for size exclusion chromatography, several hours for dialysis, and about one day for electrophoresis gel extraction.

As a conventional technique related to the separation and purification method of labeled nucleic acid, Japanese Unexamined Patent Publication (Kokai) No. 2003-511046 discloses a method in which a plurality of dye-labeled polynucleotides and dye-labeled molecules (undetected dye-labeled molecules) not attached to the polynucleotide Is contacted with a plurality of particles comprising a hydrophilic crosslinked tertiary polymer matrix containing therein a hydrophobic porous adsorbent material capable of adsorbing undetected dye label molecules, and the undetected dye label molecules are immobilized on a hydrophilic matrix To the hydrophobic substance, and the dye-labeled polynucleotide does not pass through the hydrophilic matrix due to its size, thereby removing undetected dye-labeled molecules from the mixture.

However, the method has disadvantages such as inconvenience in separation process and long time, especially separation purification time is long, and the technology for separation and purification of the nucleic acid variant developed so far is difficult to apply to a large-scale process There is a problem.

In addition, in JP-A-10-1446316, regarding the high-efficiency separation and purification of chemically-labeled nucleic acids, the hydrophobicity of the marker and the hydrophilic nature of the nucleic acid cause unreacted markers and labeled nucleic acids to have different solubilities in water and organic solvents Discloses a method capable of separating and purifying a labeled nucleic acid from a mixture containing an unreacted marker and a labeled nucleic acid with high efficiency by using the property of the labeled nucleic acid, Although the method of separating and removing the unreacted hydrophobic marker into the organic solvent phase by using the difference in solubility in the solvent is used, this method has a limitation that the marker should exhibit the hydrophobic property.

Therefore, there is a continuing need to develop a technique for separating and removing unreacted markers more efficiently according to the inherent characteristics of the markers regardless of the hydrophilic or hydrophobic properties of the markers, and a kit using the same .

The present invention is a simple and efficient method for efficiently separating and purifying only the labeled nucleic acid from a mixture containing a chemical marker and a nucleic acid capable of reacting with the chemical marker and a nucleic acid labeled with a non-reactant, And provides a method that can be used universally.

The present invention also relates to a kit that can be used for the separation and purification of labeled nucleic acids using the above separation and purification method.

In order to achieve the above object, the present invention provides a method for preparing a nucleic acid comprising the steps of: a) preparing a reaction solution by reacting a chemical marker and a nucleic acid capable of reacting with the chemical marker in an aqueous solution; b) adjusting the pH of the reaction solution to within a predetermined range; c) adding an organic solvent separated into the aqueous solution layer and the organic solvent layer to the pH-adjusted reaction solution, and stirring to prepare a mixed solution; And d) removing at least a portion of the organic solvent.

In one embodiment, the organic solvent added in the step c) may include water (H2O).

In one embodiment, the organic solvent may be an organic solvent in which water (H20) is saturated.

In one embodiment, in the step b), the pH of the predetermined reaction solution may be in a range lower than the pKa of the chemical marker, and in this case, the step b) may be carried out by mixing the aqueous solution adjusted to pH lower than the chemical marker pKa, By mixing the solution, the pH of the mixed reaction solution can be adjusted within a predetermined range.

In one embodiment, e) further comprises performing the steps b) to d) one or more times.

In one embodiment, the chemical marker may be a hydrophobic fluorescent dye or a hydrophilic fluorescent dye.

In one embodiment, the nucleic acid may be an oligonucleotide.

In one embodiment, the chemical marker may be at least one selected from Alexa 488, Cy 5, ATTO 633, Cy 3, Cy 3B, ATTO 550, ATTO 390, ATTO 647N, Rhodamine 6G and Nile red.

In one embodiment, the organic solvent may be an organic solvent having a log P value of 0.6 to 4.0.

In one embodiment, the organic solvent is selected from the group consisting of alcohols having 3 to 8 carbon atoms, ether having 3 to 8 carbon atoms, chloroform, alkyl acetates having 2 to 6 carbon atoms, aromatic hydrocarbons having 6 to 12 carbon atoms, linear or branched An alkane, a cycloalkane having 5 to 14 carbon atoms, a linear or branched alkyl group having 5 to 12 carbon atoms, a hydrocarbon having a cycloalkyl group having 5 to 12 carbon atoms, a 1,1,1-trichloroethane or a mixture thereof Lt; / RTI >

In one embodiment, it may include centrifuging the mixed solution between steps c) and d).

According to an embodiment of the present invention, there is provided a method of detecting a nucleic acid, comprising the steps of: (a) providing a first container containing a chemical marker capable of reacting with a nucleic acid and capable of reacting with the nucleic acid; A third container containing an acidic aqueous solution having a predetermined pH value; And a fourth container containing an organic solvent containing at least a part of water (H2O), wherein the pH of the acidic aqueous solution in the third container is such that the chemical indicator in the first container reacts with the nucleic acid, Wherein the pH of the mixed solution obtained by mixing the acidic aqueous solution of the third container is set to be lower than the pKa of the chemical marker.

In another embodiment of the present invention, the present invention provides a pharmaceutical composition comprising a first container comprising a chemical marker capable of reacting with a nucleic acid; A second container for reacting the nucleic acid with a chemical marker in the first container; A third container containing an acidic aqueous solution having a predetermined pH value; Wherein the pH of the acidic aqueous solution in the third container is such that the chemical markers in the first container are in contact with the nucleic acid in the second container, Wherein the pH of the mixed solution obtained by mixing the acidic aqueous solution of the third container after the reaction is set to be lower than the pKa of the chemical markers.

The present invention also relates to a pharmaceutical composition comprising a first container comprising a chemical marker capable of reacting with a nucleic acid; A second container for reacting the nucleic acid with a chemical marker in the first container; A third container containing an acidic aqueous solution having a predetermined pH value; And an organic solvent containing at least a part of water (H2O); Wherein the pH of the acidic aqueous solution in the third container is such that the chemical indicator in the first container reacts with the nucleic acid in the second container, Wherein the pH of the mixed solution obtained by mixing the acidic aqueous solution of the third container is set to be lower than the pKa of the chemical marker.

In this case, the organic solvent in the kit for separating and purifying the labeled nucleic acid according to the present invention may contain water in a saturated state.

The kit for separation and purification of the labeled nucleic acid may further comprise a container containing a basic aqueous solution for adjusting the pH of the acidic aqueous solution of the third container after the chemical marker and the nucleic acid are reacted, , And a separation device capable of separating the organic solvent and the aqueous solution.

According to the present invention, the neutral state of the marker used for labeling is induced by controlling the pH of the reaction mixture aqueous solution according to the reaction between the nucleic acid and the chemical marker irrespective of the hydrophilic and hydrophobic properties of the fluorescent substance (Dye) used as a chemical marker By increasing the solubility in the organic solvent layer, unreacted markers can be easily separated and removed.

That is, when a fluorescent substance (Dye) used as a chemical marker is released by proton ions and then ionized, the pH is controlled to a range lower than the inherent pKa value, so that various kinds of unreacted markers can be efficiently A novel method capable of easily separating and purifying a labeled nucleic acid, and a kit using the same.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing changes in the absorbance of the respective materials according to the pH range (pH = 1.8 to 9) of commonly used chemical markers. FIG.

2 is a cross- FIG. 7 is a conceptual diagram illustrating a method for removing unreacted dyes from a reaction with a nucleic acid and a chemical marker for preparing labeled oligonucleic acid through pH control of an aqueous solution according to the present invention.

FIG. 3 is a graph showing the relationship between the amount of nucleic acid (DNA) and the amount of nucleic acid (nucleic acid) added to the lower aqueous solution layer and the upper organic solvent layer under constant PH conditions according to Examples (Examples 1, 2, ) And the degree of distribution of the fluorescent dye.

FIG. 4A is a graph showing the results obtained by treating an organic solvent under pH = 8.5 conditions according to Comparative Example 1 of the present invention, separating it into an aqueous solution and an organic solvent, separating the fluorescent dye (Cy 5) ) Was treated with an organic solvent.

FIG. 4 (B) is a graph showing the relationship between the fluorescence dye (Cy 5) and the nucleic acid (DNA) remaining after being dispersed in an aqueous solution and an organic solvent after being treated with an organic solvent under pH = 3 according to Example 1 of the present invention, Is a graph showing the concentration of the organic solvent treated in each step.

FIG. 5 is a graph showing the relationship between the concentration of the mixture (Cy (1)) in the aqueous solution separated and purified by the conventional ethanol recrystallization method (upper graph in FIG. 5) and the solvent extraction method 5 and DNA) were separated by liquid chromatography (HPLC), respectively.

FIG. 6A is a graph showing the relationship between the amount of the aqueous solution separated and purified by the ethanol recrystallization method (upper left graph in FIG. 6) and the solvent extraction method using the pH control according to the first embodiment (upper right graph in FIG. 6) (Cy5 and dsDNA) were obtained through confocal microscopy.

FIG. 6B is a graph showing the relationship between the amount of the aqueous solution separated and purified by the ethanol recrystallization method (lower left graph in FIG. 6) and the solvent extraction method using the pH control according to Example 1 (lower right graph in FIG. 6) (Cy 5 and dsDNA) on the photoreceptor of the present invention.

FIG. 7A is a graph showing the absorbance of Cy3B remaining in the aqueous solution according to the pH condition after the unreacted Cy3B fluorescent dye is separated using the pH controlled separation and purification method according to Example 4 of the present invention. FIG.

7B is a graph showing the concentration of Cy3B remaining in the aqueous solution after each of the unreacted Cy3B fluorescent dyes was separated under pH = 2 conditions according to Example 4 of the present invention, to be.

(B) ssDNA at pH 8.5, (C) ssDNA at pH 1.6 at 38 ° C for 2 hours (pH 3.0), (b) And (D) high performance liquid chromatography (HPLC) of samples containing guanine and adenine.

Hereinafter, the present invention will be described in detail. But may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

In addition, the present invention is capable of various modifications and various forms, and specific embodiments are illustrated in the drawings and described in detail in the following description. It is to be understood, however, that the invention is not intended to be limited to the particular forms disclosed, but on the contrary, is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Also, in the present application, the terms " comprises ", or " having ", etc. are intended to specify that there are performed features, numbers, steps, operations, elements, But do not preclude the presence or addition of one or more other features, integers, steps, operations, components, parts, or combinations thereof.

A method of separating and purifying a chemically-labeled nucleic acid according to the present invention comprises the steps of: a) preparing a reaction solution by reacting a chemical marker and a nucleic acid capable of reacting with the chemical marker in an aqueous solution; b) adjusting the pH of the reaction solution to within a predetermined range; c) adding an organic solvent separated into the aqueous solution layer and the organic solvent layer to the pH-adjusted reaction solution, and stirring to prepare a mixed solution; And d) removing at least a portion of the organic solvent.

Hereinafter, the respective steps of the method for separating and purifying nucleic acids proposed in the present invention will be described in detail.

In step (a) of the present invention, the labeling reaction of the chemical marker and the nucleic acid capable of reacting with the chemical marker can be carried out under standard conditions generally known in aqueous solution.

In this case, the reaction time and the reaction temperature are not limited to a specific range. For example, the reaction may be performed in the range of 0 to 50 degrees for 5 minutes to 12 hours. For example, For 2 to 3 hours.

Here, the chemical marker is excessively more than the number of moles of the nucleic acid in that it minimizes the unreacted nucleic acid after the reaction. For example, the chemical marker is at least 1 time, preferably at least 2 times, more preferably at least 5 times, More preferably 10 times or more, and it is preferably 5 times or more in view of the cost of the chemical marker and the subsequent purification process.

The chemical markers used in the present invention are used as markers for the detection of nucleic acids in the art and include optical sensing materials that can be detected through ultraviolet, infrared or visible light, or can be detected through a change in refractive index, Or electrically detectable material, and typically a fluorescent dye can be used.

The fluorescent dyes may be generally divided into hydrophilic fluorescent dyes and hydrophobic fluorescent dyes. All of these hydrophilic fluorescent dyes or hydrophobic fluorescent dyes are substituted with a substituent which is removed when reacting with the nucleophile of the nucleic acid so as to easily react with the nucleic acid. And a leaving group.

Examples of fluorescent dyes that can be used as the chemical markers include Acridine homodimer and derivatives thereof, Acridine Orange and derivatives thereof, 7-aminoactinomycin D , 7-AAD) and derivatives thereof, Actinomycin D and derivatives thereof, ACMA (9-amino-6-chloro-2-methoxyacridine and derivatives thereof, DAPI) Ethidium bromide and derivatives thereof, ethidium homodimer-1 (EthD-1) and derivatives thereof, ethidium homodimer-2 (EthD-2) and derivatives thereof, dihydroethidium and derivatives thereof, ethidium bromide and derivatives thereof, And derivatives thereof, Ethidium monoazide and derivatives thereof, Hexidium iodide and derivatives thereof, bisbenzimide (Hoechst 33258) and derivatives thereof, Hoechst 33342 ) And its oil Hydroxystilbamidine and derivatives thereof, LDS 751 and derivatives thereof, Propidium Iodide (PI), and derivatives thereof, as well as derivatives thereof, Cy3, Cy3B, ATTO550, ATTO39O, ATTO647N, and ATTO647N. The chemical markers may be selected from the group consisting of Ala, Rhodamine 6G, and Nile red.

On the other hand, labeling using a fluorescent chromophore dye is preferred as the chemical marker, but it is apparent that other forms of detectable labeling are also available to those skilled in the art. For example, microparticles, gold nanoparticles (Reichert et al., Anal. Chem. 72: 6025-6029, 2000) and microbeads (Lacoste et al. , Proc. Natl. Acad. Sci. USA 97 (17): 9461-9466, 2000) can be used either as negative chargeable ligands or as positively charged ligands, so that surface charge control at non- .

In the present invention, a nucleic acid capable of reacting with a chemical marker in the present invention is a water-soluble polymer including a sugar, a phosphoric acid and a base, and it means DNA or RNA or a mixture thereof. In the present invention, the nucleic acid means a nucleic acid having more nucleotides than the oligonucleic acid and oligonucleotide Lt; RTI ID = 0.0 > polynucleotides < / RTI > Herein, the oligonucleotide means that the number of nucleotides is 100 or less or 50 or less.

In step a) of the present invention, the nucleic acid is pre-treated so as to bind to a chemical marker, which is obtained by introducing a functional group capable of reacting with a chemical marker into the nucleic acid, or by commercially purchasing a nucleic acid already having a functional group introduced thereto .

Generally, as a method for binding a nucleic acid with a marker, a nucleic acid having a nucleophilic functional group such as a primary amine group or a thiol group is reacted with a marker such as a fluorescent dye or a probe functionalized with an electrophilic functional group to produce a labeled nucleic acid , And the functional groups and markers of the nucleic acid can be linked through other linkages.

Here, if the chemical marker and the nucleic acid can be combined through the reaction with the chemical marker, the kind of the functional group present or newly attached to the nucleic acid is not limited. However, considering the reactivity with the chemical marker, (Functional group) can be selected. For example, the nucleophilic functional group includes a primary amine group or a thiol group. For example, when the functional group of the nucleic acid is a primary amine group, the chemical marker is an ester, isothiocyanate, isocyanate, acyl azide, NHS ester , Sulfonyl chloride, aldehyde, and the like. When the functional group of the nucleic acid is a thiol group, the label may include functional groups such as haloacetyl, alkyl halide derivatives, maleimide, aziridine, etc., A primary amine group may be preferably used.

The method for producing a nucleic acid capable of binding to a chemical marker by attaching such a functional group to a nucleic acid can be carried out by a conventional method in the art besides the above exemplified method.

The step b) of the present invention is a step of adjusting the pH of the reaction solution obtained by the reaction of the chemical marker with the nucleic acid to a predetermined range, thereby changing the ionization state of the chemical marker, When the organic layer and the aqueous solution layer are separated from each other by mixing the organic solvent in c), the unreacted chemical markers can be effectively removed according to the difference in solubility between the aqueous solution and the organic solvent. do.

The pH range of the reaction solution usually used in the present invention may be in the range of pH 1 to pH 10, preferably in the range of pH 1 to pH 7, more preferably in the range of pH 2 to pH 5 .

On the other hand, in the step b), it is preferable that the pH of the predetermined reaction solution is maintained in a range lower than the pKa of the chemical marker. Typically, in the case of the above-mentioned chemical markers, a substance which is weakly acidic in aqueous solution and which is dissociated into an anion portion of a chemical marker and a proton (H ⁺ ) can be used, wherein the marker has a neutral and anionic concentration The ratio follows the Henderson-Hasselbalch equation, and when pH = pKa as shown in Equation 1 below, the concentrations of anionic and non-ionic types become equal.

[Equation 1] pH = pKa + log10 ( [A -] / [HA])

When the pH of the mixture solution of the reaction solution containing unreacted chemical markers is greater than the pKa value of the chemical markers according to the above formula 1, the concentration of the ionic chemical markers having a negative charge becomes higher than that of the neutral markers As a result, the ionic indicator becomes more soluble in the aqueous solution, and therefore, the unreacted marker can not be extracted and separated into the organic solvent layer in an aqueous solution of pH higher than the pKa value of the chemical marker.

Conversely, when the pH of the mixture solution of the reaction solution containing unreacted chemical markers is less than the pKa value of the chemical markers according to the above formula 1, the concentration of neutral chemical markers is dominant, and the chemical markers of such neutral states It becomes possible to have a higher solubility in the organic solvent phase.

By using this principle, an unreacted neutral chemical marker is extracted and separated into an organic solvent layer by mixing a certain amount of an organic solvent with an aqueous solution of the mixture containing a nucleic acid and an unreacted chemical marker and then inducing the layer separation It is possible to remove unreacted chemical markers more efficiently and easily.

Therefore, in the present invention, by using a principle of extracting and separating a neutral chemical marker existing at a pH lower than the pKa value of a chemical marker by an organic solvent, it is possible to use both a hydrophobic chemical marker and a hydrophilic chemical marker as chemical markers .

In order to adjust the pH of the reaction solution, an acidic solution having a predetermined concentration may be used. Preferably, HNO 3, HClO 4, H 2 SO 4, HCl, HBr, HI and the like, which are classified as strong acids, have.

At this time, in order to prevent an accident caused by an exothermic reaction due to a base reaction in the production of the acidic solution, it is preferable to add the aqueous solution acid in small quantities under a condition of a large amount of aqueous solution through a mechanism such as a pipette.

That is, in the step b) of the present invention, the pH of the mixed reaction solution can be adjusted to a predetermined range by mixing the reaction solution with an aqueous solution adjusted to pH lower than the pKa of the chemical marker, An acidic solution can be used to prepare an aqueous solution adjusted to a pH lower than the pKa.

The stirring method for adding the reaction solution to the aqueous solution adjusted to a pH lower than the pKa of the chemical marker and for increasing the mixing time and the mixing efficiency when mixing is not limited and it is also possible to use a stirring device such as simple stirring or shaking or using a vortex mixer .

Meanwhile, in the present invention, in order to adjust the pH conditions of the acid solution or the mixed solution of the acid solution and the reaction solution, the pH condition of the present invention may be adjusted by using a commercially available pH meter or using a pH test paper having a measurement range of 1 to 14 .

The pKa value of the chemical marker can be measured using a potential difference method and ultraviolet / visible light spectroscopy. FIG. 1 is a graph showing the change in absorbance of each of the materials according to the pH range (pH = 1.8 to 9) of commonly used chemical markers. In the present invention, pH = 1.8 to 9.0 under ultraviolet / visible ray spectroscopy 1, where the absorbance of the fluorescent dye is 1/2, the pH = pKa condition is established in the Henderson-Hasselbalch equation as the concentrations of the anion-state marker and the neutral-state marker become the same, The pKa value of each fluorescent dye can be experimentally obtained.

This is because the acid dissociation constant (pKa) is dependent on the temperature dependence when the dissociation reaction of the acid is an exothermic reaction and the endothermic reaction. Specifically, when the dissociation reaction is an endothermic reaction, the pKa value decreases as the temperature increases, and when the dissociation reaction occurs as an exothermic reaction, the pKa value increases as the temperature increases.

Meanwhile, in the step c) of the present invention, an organic solvent separated into an aqueous solution layer and an organic solvent layer is added to a reaction solution whose pH is controlled, and stirred to prepare a mixed solution. Although the amount of the organic solvent is not limited, it is preferable that the volume of the organic solvent is excessively larger than the volume of the mixed aqueous solution of the step b) in order to remove the unreacted markers earlier and shorten the purification time by reducing the number of purification. For example, the organic solvent may be added at a volume of at least 2 times, at least 3 times, at least 5 times, or at least 10 times.

In addition, the organic solvent used in step c) may include water (H2O) in the organic solvent, and preferably the organic solvent is an organic solvent in which water (H2O) is saturated desirable.

Here, the "organic solvent saturated with water" refers to an organic solvent in which the organic solvent dissolves as much as possible to dissolve the water so that the dissolution rate has reached equilibrium without further dissolving the water. Here, distilled water can be preferably used to minimize the incidental effect of the water used to saturate the organic solvent.

On the other hand, when the organic solvent is used in a state of being not saturated with water, in the process of adding and stirring an organic solvent, a part of water in the water layer can be dissolved in the organic solvent layer, Can occur.

When the organic solvent is added in step c), the organic solvent is added in excess of the volume of the mixed aqueous solution in step b). Therefore, when water is not contained in the organic solvent, It is disadvantageous that the water layer containing the chemically labeled nucleic acid and the organic solvent layer containing the unreacted label may not be separated from each other or the aqueous solution layer may be difficult to be separated or separated.

Therefore, when an organic solvent containing water or an organic solvent saturated with water is used, when an organic solvent is added and stirred, water which should be present in the receptive layer due to layer separation is further dissolved in the organic solvent layer By not migrating, there is an advantage that it does not affect the concentration of the labeled nucleic acid present in the aqueous solution layer.

Meanwhile, the stirring time in the step c) is not limited, and may range from several seconds to several hours. Most of the time is sufficient for several seconds to several minutes, and the stirring method thereof is also not limited, Shaking, or using a device such as a vortex mixer.

In the meantime, the present invention utilizes the difference in solubility between water and an organic solvent of a chemical marker at a preset pH, and it is an object of the present invention to provide an organic solvent having a distribution coefficient of chemical markers of 2 or more at a pH range Can be preferably used.

[Equation 2] Distribution coefficient = [Corganic] / [Cwater]

(Wherein [Corganic] is the molar concentration of the chemical marker in the organic solvent and [Cwater] is the molar concentration of the chemical marker in the water).

Herein, the partition coefficient is a ratio of the concentration of CA to the concentration of CB in each solution when the substance dissolves in equilibrium at a certain temperature and pressure in two unmixed liquids A and B, that is, K = CA / CB, and the partition coefficient can be obtained by measuring the concentration of a chemical marker in an organic solvent and the concentration of a chemical marker in water via a UV / Vis spectrometer (sptectrometer).

The partition coefficient is an index showing the solubility of a solvent in water and an organic solvent. In the present invention, a correlation between a chemical marker used for separation and purification of a chemically labeled nucleic acid and an organic solvent, The kind of the chemical marker and the organic solvent are not limited.

In the present invention, the chemical markers in the neutral state have a partition coefficient of 2 or more, preferably 10 or more, more preferably 25 or more, still more preferably 50 or more, still more preferably 100 or more, , More preferably 200 or more, and more preferably 300 or more. The larger the partition coefficient, the easier separation of the labeled nucleic acid from the unreacted marker.

That is, the value of the partition coefficient varies depending on a constant pH condition. FIG. 3 shows the distribution of fluorescent dyes between the nucleic acid layer and the aqueous solution layer and hexane as the organic solvent, The distribution of the fluorescent dye to the organic solvent layer is increased under the low pH condition.

The organic solvent used in the present invention is not particularly limited as long as it is an organic solvent from which water and a layer can be separated and in which an aqueous solution layer and an organic solvent layer can be separated.

When the Log P value of the organic solvent is less than 0.6, the water and the organic solvent layer are not separated well. Therefore, an organic solvent having a log P value of 0.6 or more may be used in the present invention.

Here, the log P value means the log value of P, which means the partition coefficient of the organic solvent for the same molar mixture of octanol and water.

On the other hand, the upper limit of the log P value of the organic solvent is not limited, but if the log P value is too high, the chemical markers of the neutral state may be dissolved in the water rather well, so that the chemical markers of neutral state and the organic solvent 2 is satisfied, it is possible to select an appropriate organic solvent.

Organic solvents which can be preferably used in relation to chemical markers in general neutral state are organic solvents having a log P value in the range of 0.6 to 4.0, more preferably organic solvents having a log P value in the range of 0.6 to 3.5, More preferably, it is an organic solvent having a log P value in the range of 0.6 to 3.0.

Examples of such organic solvents having a log P value of 0.6 to 4.0 include alcohols having 3 to 8 carbon atoms, ethers having 3 to 8 carbon atoms, chloroform, alkyl acetates having 2 to 6 carbon atoms, aromatic hydrocarbons having 6 to 12 carbon atoms, A linear or branched alkane having 1 to 14 carbon atoms, a cycloalkane having 5 to 14 carbon atoms, a linear or branched alkyl group having 5 to 12 carbon atoms and a cycloalkyl group having 5 to 12 carbon atoms, 1,1,1-trichloroethane 1-butanol, isoamyl alcohol and pentanol; alcohols having 4 to 8 carbon atoms, such as ethyl acetate, diethyl ether, diisopropyl ether, butyl acetate, chloroform, Benzene, 1,1,1-trichloroethane, toluene, hexene and the like can be used.

Step d) of the last step in the present invention is a step of removing at least a part of the organic solvent, at least partially removing an organic solvent mainly comprising unreacted chemical markers, preferably in a layered organic layer Most organic solvents can be removed. As a method for removing the organic solvent, a commonly used method can be used. In the continuous process, a decanter is used. In the batch process, a separating funnel or the like can be used. When the amount is small, Can be removed.

On the other hand, when the step of centrifuging the mixed solution is further included between the step c) and the step d), it may be advantageous in terms of shortening the purification time and increasing the purification yield.

The centrifugation method is not particularly limited, and can be performed by setting appropriate centrifugation conditions using a centrifuge. For example, 500 g, 1000 g, 2000 g, 4000 g or 6000 g, and the centrifugation time can be 10 seconds, 20 seconds, 30 seconds, or about 1 minute, Phase separation occurs in a short time depending on the difference in density of the mixed aqueous solution and the organic solvent, and the organic solvent can be removed more efficiently in the step d).

On the other hand, when the steps b) to d) are sequentially performed in the respective steps of the present invention, the removal rate of unreacted markers is increased, so that b) and d) .

In addition, if necessary, in the method for removing unreacted chemical markers according to the present invention, the step of removing the unreacted nucleic acid may be further performed before the step b) or after the step d).

In this regard, when the nucleic acid having a functional group is reacted with an excessive amount of a chemical marker, the step of removing the unreacted nucleic acid remaining in the aqueous solution layer is almost meaningless since the reaction efficiency between the nucleic acid and the marker is considerably high. For example, when a high quality amine-modified oligonucleic acid or the like is used to react with a marker, the reaction efficiency between the nucleic acid and the marker is considerably high, and the step of further removing the unreacted nucleic acid remaining in the aqueous solution layer is almost meaningless In the case where the reaction efficiency between the nucleic acid and the marker is not high, in order to selectively purify only the labeled nucleic acid, the unreacted nucleic acid remaining in the aqueous solution layer is further removed And the method of removing such unreacted nucleic acid in the present invention can be carried out according to a known method.

Generally, unreacted nucleic acid can be removed by chromatography such as reversed phase and ion exchange, or gel electrophoresis. Since the nucleic acid labeled with a hydrophobic marker is partially hydrophobic, the hydrophilic nature of the unreacted nucleic acid And thus simpler purification is possible. Thus, the labeled nucleic acid and the unreacted nucleic acid can be separated from each other by HPLC reverse phase chromatography, and can be purified using a disposable reversed phase column (Polypak, Glen Research, Inc., USA).

2 is a cross- The present invention relates to a method of separating and purifying a chemically labeled nucleic acid according to the present invention in a temporal order, and more particularly, to a method for producing a labeled nucleic acid A method for removing unreacted dyes from a reaction between a marker and nucleic acid is exemplarily shown.

As shown in FIG. 2, the method for separating and purifying nucleic acid according to the present invention comprises the steps of adding a pH-adjusted aqueous solution to a labeled nucleic acid reaction solution, and then adding 2 to 4 When butanol saturated with boiling water was additionally added, stirring was performed using a vortex mixer for 10 seconds or more, and then a butanol solution in which an unreacted marker located at the upper part was dispersed was removed by using a micropipette or the like, It is schematically shown that an aqueous solution in which a highly purified labeled nucleic acid is dispersed can be obtained.

In addition, the present invention can provide a kit for a method for separating and purifying a chemically-labeled nucleic acid using the above method.

In an embodiment of the kit, the kit includes a chemical label capable of reacting with a nucleic acid, and a first container in which, when a nucleic acid containing an aqueous solution is introduced, the nucleic acid and the chemical marker can react with each other; A third container containing an acidic aqueous solution having a predetermined pH value; And a fourth container containing an organic solvent containing at least a part of water (H2O), wherein the pH of the acidic aqueous solution in the third container is such that the chemical indicator in the first container reacts with the nucleic acid, And the pH of the mixed solution obtained by mixing the acidic aqueous solution of the third container is set to be lower than the pKa of the chemical marker. In this case, the kit may be prepared such that after the nucleic acid and the chemical marker react in the first container, the acidic aqueous solution is added to the third container, the pH is adjusted, and then the organic solvent in the fourth container is introduced, The nucleic acid can be purified.

In another embodiment, the kit comprises a first container comprising a chemical marker capable of reacting with a nucleic acid; A second container for reacting the nucleic acid with a chemical marker in the first container; A third container containing an acidic aqueous solution having a predetermined pH value; Wherein the pH of the acidic aqueous solution in the third container is such that the chemical markers in the first container are in contact with the nucleic acid in the second container, The pH of the mixed solution obtained by mixing the acidic aqueous solution of the third container after the reaction is set to be lower than the pKa of the chemical marker. In this case, the kit may be prepared such that after the nucleic acid and the chemical marker react in the second container, the acidic aqueous solution is added to the third container, the pH is adjusted, and then the organic solvent in the fourth container is introduced, The nucleic acid can be purified.

In another embodiment, the kit comprises a first container comprising a chemical marker capable of reacting with a nucleic acid; A second container for reacting the nucleic acid with a chemical marker in the first container; A third container containing an acidic aqueous solution having a predetermined pH value; And an organic solvent containing at least a part of water (H2O); Wherein the pH of the acidic aqueous solution in the third container is such that the chemical indicator in the first container reacts with the nucleic acid in the second container, And the pH of the mixed solution obtained by mixing the acidic aqueous solution of the third container is set to be lower than the pKa of the chemical marker. In this case, the kit can be prepared by reacting a chemical marker in the first container with a nucleic acid in the fifth container in a second container, and then introducing an acidic aqueous solution in the third container to adjust the pH, The labeled nucleic acid can be purified.

The kit for separating and purifying the labeled nucleic acid according to the present invention may further comprise a container containing a basic aqueous solution for adjusting the pH in mixing the acidic aqueous solution of the third container with the nucleic acid after the chemical label is reacted with the nucleic acid, . In addition, the kit may further include a separation device capable of separating the organic solvent and the aqueous solution, and a separating funnel, a pipette, a centrifuge, etc. may be used as an exemplary separation device.

A kit for separating and purifying a labeled nucleic acid according to the present invention is a device for actually applying a method for separation and purification of a labeled nucleic acid as described in detail above, wherein the materials of the first to fourth containers are metal, glass, rubber, Materials and the like can be used, but the present invention is not limited thereto, and the size thereof can be variously modified in accordance with the content of the organic solvent and the nucleic acid to be used, and can be appropriately changed by a person skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent to those skilled in the art, however, that these examples are provided to further illustrate the present invention, and the scope of the present invention is not limited thereto.

≪ Example 1 >

1) A modified oligo nucleic acid (DNA 30 mer / 60mer) having an amine group and a fluorescent dye Cy 5 (Cy5 NHS ester # PA 15104) in a 2 ml tube were reacted at a molar ratio of 1:10 and maintained at room temperature for 2 to 3 hours .

- Cy5 dissolved in dimethyl sulfoxide (DMSO): 25 nmole / 5.21 ul

- Nucleic acid (DNA 30 mer / 60 mer) dissolved in water: 5 nmole / 8.17 ul

- 0.2 M SB (Sodium Borate) buffer (pH 8.5): 20 ul

- Water: 16.62 ul

- total reaction solution volume: 50 ul

The sequence of the modified oligonucleotide (DNA 30 mer, 60mer) is shown in Table 1 below.

[Table 1]

2) Add a certain amount of HCl solution to make 500 μl of the aqueous solution adjusted to pH 3.0, and then add the solution of reaction 1.

3) To the solution prepared in 2 was added 1.5 ml of n-butanol saturated with water, mixed with a vortex mixer for about 10 seconds, and centrifuged at 4000 g for about 10 seconds.

4) The organic solvent layer as the upper layer of the solution layer divided by 3 was removed by pipette.

5) The procedures 2 to 4 were repeated two more times, and the time taken to complete the above steps 2 to 4 was within 3 minutes.

≪ Example 2 >

The procedure of Example 1 was repeated except that Cy 5 was changed to Cy 3 (GE healthcare #PA 13106) as a fluorescent dye and the aqueous solution in Step 2 was adjusted to pH = 2.

≪ Example 3 >

Except that Cy 5 was changed to Alexa 488 (Alexa Fluor 488 NHS Ester (Succinimidyl Ester) Thermo Fisher scientific A 20000) as a fluorescent dye, and the aqueous solution of step 2 was adjusted to pH = 2 .

<Example 4>

The procedure of Example 1 was repeated except that Cy 5 was changed to Cy 3B (GE healthcare #PA 63100) as a fluorescent dye and the aqueous solution of Step 2 was adjusted to pH = 2.

&Lt; Comparative Example 1 &

1) A modified oligo nucleic acid (DNA 30 mer / 60mer) having an amine group in a 2 ml tube was reacted with a fluorescent dye Cy 5 at a reaction ratio of 1:10 and maintained at room temperature for 2 to 3 hours.

- Cy5 dissolved in dimethyl sulfoxide (DMSO): 25 nmole / 5.21 ul

- Nucleic acid (DNA 30 mer, 60mer) dissolved in water: 5 nmole / 8.17 ul

- 0.2 M SB (Sodium Borate) buffer (pH 8.5): 20 ul

- Water: 16.62 ul

- total reaction solution volume: 50 ul

2) Add a certain amount of NaOH solution to make 500 μl of an aqueous solution having a pH value of 8.5, and then add the solution to which the reaction 1 has been passed.

3) To the solution prepared in 2 was added 1.5 ml of n-butanol saturated with water, mixed with a vortex mixer for about 10 seconds, and centrifuged at 4000 g for about 10 seconds.

4) The organic solvent layer as the upper layer of the solution layer divided by 3 was removed by pipette.

5) The procedures 2 to 4 were repeated two more times, and the time taken to complete the above steps 2 to 4 was within 3 minutes.

&Lt; Comparative Example 2 &

The procedure of Example 1 was repeated except that Cy 5 was changed to Cy 3 as the fluorescent dye and the aqueous solution of Step 2 was adjusted to pH = 8.5.

&Lt; Comparative Example 3 &

The procedure of Example 1 was repeated except that Cy 5 was changed to Alexa 488 as the fluorescent dye and the aqueous solution of Step 2 was adjusted to pH = 8.5.

Fig. 3 is a graph showing the relationship between the amount of the nucleic acid and the fluorescence (fluorescence) of the organic solvent layer on the lower aqueous solution layer and the upper organic solvent layer under constant PH conditions according to the examples (Examples 1, 2 and 3) (A) Cy5 (Comparative Example 1 on the left, Example 1 on the right), (B) Cy3 (Comparative Example 2 on the left, Comparative Example 2 on the right), Cy3 (Example 2), (C) Alexa 488 (Comparative Example 3 on the left, and Example 3 on the right).

According to FIG. 3, according to actual photographs showing the distribution of nucleic acid and fluorescent dye to the lower aqueous solution layer and the upper organic solvent layer under pH = 8.5 and pH = 3 and 2 conditions, The degree of distribution of the fluorescent dye can be visually confirmed.

More specifically, as a result of Example 1, it can be observed that Cy5 is dissolved in an anionic state in the lower aqueous solution under the condition of pH = 8.5 as shown in Fig. 3 (A) 5 is converted to a neutral state in combination with the proton, and is extracted and dissolved in the upper organic solvent layer. At this time, the fluorescence present in the lower part may be a nucleic acid labeled with a fluorescent dye or an unreacted unreacted fluorescent dye. This pattern appears in Fig. 3 (B) Cy 3 according to the results of Example 2 and is the same in Fig. 3 (C) Alexa 488 in the result of Example 3.

Fig. 4A is a graph showing the relationship between the concentration of the fluorescent dye (Cy5) remaining in the aqueous solution and the concentration of the nucleic acid after being separated into an aqueous solution and an organic solvent after being treated with an organic solvent under the condition of pH = 8.5 according to Comparative Example 1 of the present invention FIG. 4 (B) is a graph showing the results obtained by treating an organic solvent under pH = 3 according to Example 1 according to the present invention, dividing it into an aqueous solution and an organic solvent, (Cy 5) and the concentration of the nucleic acid in each step of treating the organic solvent.

As a result of Example 1 according to FIG. 4, when the Cy 5 fluorescent dye was repeated several times by the purification method of the present invention under the condition of pH = 3, Cy 5 and DNA concentrations were found to be almost similar in the aqueous solution, Since the concentration of Cy5 and DNA is obtained from Cy5-labeled DNA obtained through a one-to-one reaction, Cy5, an unreacted fluorescent dye, is almost completely removed on the aqueous solution.

On the other hand, in the case of separation and purification under the pH = 8.5 condition according to Comparative Example 1, Cy5 in an anionic state predominantly exists on the aqueous phase, and only Cy5 in a small amount of neutral state is distributed in the organic solvent layer to remain in the aqueous solution Cy 5 is present at a much higher concentration than DNA even when the concentration is repeated over a number of times. Since the unreacted Cy 5 appears to remain in the aqueous solution, the purification method through the pH control according to the present invention proceeds Indicating that DNA can be separated and purified with high efficiency within a short time.

FIG. 5 is a graph showing the result of separation and purification of a mixture in an aqueous solution obtained by the ethanol recrystallization method and the separation and purification method of the present invention through liquid chromatography (HPCL). More specifically, the upper graph of FIG. (Cy 5 and DNA) in aqueous solution separated and purified according to the ethanol recrystallization method according to the present invention were separated by liquid chromatography (HPLC), respectively. In the lower graph of FIG. 5, And the result of solvent extraction using pH control according to the present invention.

According to FIG. 5, the unreacted fluorescent dye Cy 5 was detected between 10 and 13 minutes in the case of the mixture purified by the ethanol recrystallization method, whereas in the case of the extraction method using pH control according to the present invention, unreacted fluorescent dye Cy 5 was not detected, indicating that the separation and purification method of the present invention is a highly efficient separation and purification method as compared with the conventional ethanol recrystallization method.

FIG. 6 shows the results of testing the fluorescent performance of the ethanol recrystallization method according to the prior art and the labeled nucleic acid isolated and purified according to the present invention.

More specifically, FIG. 6A shows the ssDNA labeled with a fluorescent dye, Cy5, together with a complementary chain, followed by confocal microscopy. As a result, 6) and the mixture (Cy5 and dsDNA) in the aqueous solution separated and purified by the solvent extraction method using the pH control according to Example 1 according to the present invention (upper right graph in FIG. 6) were examined with a confocal microscope confocal microscopy. It can be seen that there is no difference in fluorescence performance between the ethanol recrystallization method and the two samples purified by the present invention, and thus the purification method according to the present invention can be usefully used. .

FIG. 6B is a graph showing the results of separation and purification by the ethanol recrystallization method (lower left graph in FIG. 6) and the solvent extraction method using pH control according to Example 1 (lower right graph in FIG. 6) (Cy 5 and dsDNA) in the aqueous solution, the number of photons per pixel and the maximum number of photons were measured. In FIG. 6 (B), the number of photons per pixel and the maximum number of photons were measured. It can be confirmed that there is no difference in the two samples purified by the present invention. Thus, it can be seen that the fluorescence performance of the labeled nucleic acid isolated and purified through the present invention can be maintained and applied to various types of labels.

As a result of Example 4, Fig. 7 (A) shows the result of measuring the absorbance of Cy3B remaining in the aqueous solution through ultraviolet / visible light spectroscopy after separation and purification of Cy3B fluorescent dye at pH = 1.8 to 9.0. The higher the degree of separation of the unreacted Cy3B fluorescent dye, the lower the absorbance of Cy3B. 7B is a graph showing the separation and purification rate of the Cy3B fluorescent dye of the present invention under the condition of pH = 2.0.

In FIG. 8, when the samples (A) purified from the ssDNA under the conditions (pH 3.0) according to the present invention and the depurinated sample (C) treated with ssDNA at pH 1.6 and 38 ° C for 2 hours were compared, In the sample purified at pH 3.0 by the purification method according to the invention, guanine and adenine were not detected in the same manner as the sample (B) stored at pH 8.5, which is a condition for labeling nucleic acid with a fluorescent dye, In the case of using the nucleic acid separation and purification method according to the present invention, it is shown that the purified nucleic acid is destroyed by the reaction conditions or the like in the purification process according to the present invention, or the fluorescently labeled portion is not released, It is confirmed that the possibility of technical application is wide.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific descriptions are only for the preferred embodiments and that the scope of the present invention is not limited thereto Will be blind. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The simple and efficient separation and purification method of chemically labeled oligonucleic acid by pH control according to the present invention can separate and purify the labeled nucleic acid easily and efficiently with various kinds of unreacted markers regardless of the type of the marker, It is easy to manufacture a kit using the same, which is highly likely to be used in industry.

Claims (17)

1. a) preparing a reaction solution by reacting a chemical marker and a nucleic acid capable of reacting with the chemical marker in an aqueous solution;

   b) adjusting the pH of the reaction solution to within a predetermined range;

   c) adding an organic solvent separated into the aqueous solution layer and the organic solvent layer to the pH-adjusted reaction solution, and stirring to prepare a mixed solution; And

   and d) removing at least a portion of the organic solvent.
2. The method according to claim 1,

   Wherein the organic solvent added in step c) comprises water (H2O).
3. 3. The method of claim 2,

   Wherein the organic solvent is an organic solvent in which water (H2O) is saturated.
4. The method according to claim 1,

   Wherein the pH of the predetermined reaction solution is lower than the pKa of the chemical marker in the step b).
5. 5. The method of claim 4,

   Wherein the step b) comprises adjusting the pH of the mixed reaction solution to a predetermined range by mixing the reaction solution with an aqueous solution adjusted to a pH lower than the pKa of the chemical marker.
6. The method according to claim 1,

   And e) performing the steps b) to d) one or more times.
7. The method according to claim 1,

   Wherein the chemical marker is a hydrophobic fluorescent dye or a hydrophilic fluorescent dye.
8. The method according to claim 1,

   Wherein the nucleic acid is an oligonucleic acid.
9. The method according to claim 1,

   Wherein the chemical marker is at least one selected from Alexa 488, Cy 5, ATTO 633, Cy 3, Cy 3B, ATTO 550, ATTO 390, ATTO 647N, Rhodamine 6G and Nile red. .
10. The method according to claim 1,

    Wherein the organic solvent is an organic solvent having a log P value of 0.6 to 4.0.
11. The method according to claim 1,

    Wherein the organic solvent is selected from the group consisting of alcohols having 3 to 8 carbon atoms, ether having 3 to 8 carbon atoms, chloroform, alkyl acetates having 2 to 6 carbon atoms, aromatic hydrocarbons having 6 to 12 carbon atoms, linear or branched alkanes having 5 to 14 carbon atoms, A cycloalkane having 1 to 14 carbon atoms, a linear or branched alkyl group having 5 to 12 carbon atoms, a hydrocarbon having a cycloalkyl group having 5 to 12 carbon atoms, or 1,1,1-trichloroethane, or a mixture thereof A method for separating and purifying a labeled nucleic acid.
12. The method according to claim 1,

    And centrifuging the mixed solution between step c) and step d). &Lt; RTI ID = 0.0 &gt; 15. &lt; / RTI &gt;
13. A first container containing a chemical marker capable of reacting with a nucleic acid and capable of reacting with the nucleic acid and the chemical marker when a nucleic acid containing an aqueous solution is introduced;

    A third container containing an acidic aqueous solution having a predetermined pH value; And

    And a fourth container containing an organic solvent which at least partially contains water (H2O)

    The pH of the acidic aqueous solution in the third vessel may be,

    Characterized in that the pH of the mixed solution obtained by mixing the acidic aqueous solution of the third container after the chemical marker in the first container reacts with the nucleic acid is set to be lower than the pKa of the chemical marker Kit for separation and purification.
14. A first container comprising a chemical marker capable of reacting with a nucleic acid;

    A second container for reacting the nucleic acid with a chemical marker in the first container;

    A third container containing an acidic aqueous solution having a predetermined pH value; And

    And a fourth container containing an organic solvent which at least partially contains water (H2O)

    The pH of the acidic aqueous solution in the third vessel may be,

    Characterized in that the pH of the mixed solution obtained by mixing the acidic aqueous solution of the third container after the chemical marker in the first container reacts with the nucleic acid in the second container is set to be lower than the pKa of the chemical marker A kit for separating and purifying a labeled nucleic acid.
15. A first container comprising a chemical marker capable of reacting with a nucleic acid;

    A second container for reacting the nucleic acid with a chemical marker in the first container;

    A third container containing an acidic aqueous solution having a predetermined pH value; And

    A fourth container comprising an organic solvent at least partially containing water (H2O); And

    And a fifth container containing a nucleic acid capable of reacting with the chemical marker,

    The pH of the acidic aqueous solution in the third vessel may be,

    Characterized in that the pH of the mixed solution obtained by mixing the acidic aqueous solution of the third container after the chemical marker in the first container reacts with the nucleic acid in the second container is set to be lower than the pKa of the chemical marker A kit for separating and purifying a labeled nucleic acid.
16. 16. The method according to any one of claims 13 to 15,

    Wherein the kit for separation and purification of the labeled nucleic acid further comprises a container containing a basic aqueous solution for adjusting the pH in mixing the acidic aqueous solution of the third container after the chemical marker and the nucleic acid are reacted A kit for the separation and purification of labeled nucleic acids.
17. 16. The method according to any one of claims 13 to 15,

    Wherein the kit for separation and purification of the labeled nucleic acid further comprises a separation device capable of separating the organic solvent and the aqueous solution.

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