JP5815057B2 - Method for preparing sample for use in electrophoresis, electrophoresis method using the sample, and method for quantifying polynucleotide - Google Patents

Description

  The present invention relates to a method for preparing a sample to be subjected to electrophoresis, and to an electrophoresis method and a polynucleotide quantification method using the sample.

  In recent years, development of drugs composed of polynucleotides (for example, oligonucleotides) has been promoted.

  In order to effectively treat a disease using such a drug, it is necessary to accurately adjust the drug concentration in the patient's body after accurately measuring the drug concentration in the patient's body. Therefore, development of techniques for accurately quantifying polynucleotides present in various samples (for example, blood and the like) collected from patients has been underway.

  Conventionally, the LC-MS / MS method has been used for quantification of a polynucleotide contained in a sample. However, if an attempt is made to quantify a polynucleotide using mass spectrometry, multivalent ions are generated when the polynucleotide is ionized. Therefore, it is difficult to quantify a low-concentration polynucleotide by the LC-MS / MS method, and according to many literatures, a polynucleotide having a concentration of several ng / mL or more is determined by the LC-MS / MS method. Otherwise, it has been reported that quantification is not possible (see, for example, Non-Patent Document 1).

  On the other hand, the blood concentration of the drug constituted by the polynucleotide may be several ng / mL or less. Therefore, development of a highly sensitive quantitative method has been desired.

  Development of quantitative methods using nanoLC-MS / MS and quantitative methods using capillary electrophoresis combined with laser-induced fluorescence detection were advanced as highly sensitive quantitative methods.

  In the case of a quantification method using nanoLC-MS / MS, in order to separate a polynucleotide administered to a living body and a metabolite of the polynucleotide in the living body, an ion pair reagent is used as a mobile phase, and a long time is taken. There is a drawback from the viewpoint of throughput. On the other hand, in a quantitative method using capillary electrophoresis combined with laser-induced fluorescence detection, a polynucleotide administered to a living body and a metabolite of the polynucleotide can be separated in a short time.

  In other words, among the two types of quantification methods described above, the quantification method using capillary electrophoresis combined with laser-induced fluorescence detection provides more detailed data on polynucleotides in vivo in a shorter time. There is a high possibility that more appropriate treatment based on the data can be performed. Therefore, among high-sensitivity quantification methods, attention is particularly focused on the development of quantification methods using capillary electrophoresis combined with laser-induced fluorescence detection (see, for example, Non-Patent Documents 2 to 4).

  In a quantification method using capillary electrophoresis combined with laser-induced fluorescence detection, the polynucleotide is purified from a sample (for example, blood) before being subjected to capillary electrophoresis. Then, a fluorescently labeled probe that hybridizes to the polynucleotide and an internal standard substance that exhibits fluorescence (for example, a low molecular weight compound) are added to the purified polynucleotide, and then the mixture becomes a capillary. Subject to electrophoresis.

  Note that. Specific methods for performing the purification include, for example, a method of purifying a polynucleotide using a commercially available column and a method of purifying a polynucleotide by an ethanol precipitation method. Since the above purification method is a treatment method specialized for polynucleotides, the internal standard substance must be a polynucleotide. However, when a polynucleotide is used as the internal standard substance, non-specific hybridization occurs with respect to the probe or sample, and it does not function as the internal standard substance. In order to avoid this reaction, not a polynucleotide but a low molecular weight compound is used as an internal standard substance.

  The mixture during capillary electrophoresis contains nucleotides hybridized to a fluorescently labeled probe and an internal standard that exhibits fluorescence. The polynucleotide hybridized to the fluorescently labeled probe is detected by laser-induced fluorescence detection, and the amount of the polynucleotide is calculated based on the fluorescent signal derived from the detected probe. Further, the calculated amount is corrected based on the fluorescence signal derived from the internal standard substance.

  In the quantification method using capillary electrophoresis combined with laser-induced fluorescence detection, the polynucleotide contained in the sample (for example, blood) is quantified as described above.

William D van Dongen and Wilfried MA Niessen, Bioanalysis, 2011, Vol.3, p.541-564 Eunmi Ban et.al., Electrophoresis, 2013, Vol.34, p.598-604 Nasrin Khan et.al., Analytical Chemistry, 2011, Vol.83, p.6196-6201 Svetlana M Krylova et.al., Analytical Chemistry, 2010, Vol.82, p.4428-4433

  However, the conventional quantification method using capillary electrophoresis combined with laser-induced fluorescence detection is costly, requires a long time for purification, and quantifies a specific type of nucleic acid (either DNA or RNA). However, there is a problem that the recovery rate is not reflected due to lack of the internal standard substance.

  For example, as described above, in the conventional quantification method, a polynucleotide is purified from a sample (for example, blood) by a method using a commercially available column or an ethanol precipitation method before subjecting the polynucleotide to capillary electrophoresis. It is carried out.

  Therefore, the conventional quantification method has a problem that the cost is increased due to the column used for the purification and a long time is required for the purification operation.

  Further, as described above, in the conventional quantification method, the polynucleotide is purified from the sample (for example, blood) by a method using a commercially available column or an ethanol precipitation method before subjecting the polynucleotide to capillary electrophoresis. It is carried out. In this purification process, the type of polynucleotide to be purified is limited to either DNA or RNA.

  Therefore, the conventional quantification method has a problem that it is limited to quantification of a specific type of nucleic acid (either DNA or RNA).

  Further, as described above, in the conventional quantification method, when a polynucleotide is used as an internal standard substance, non-specific hybridization occurs with respect to a probe or a sample, and it does not function as an internal standard substance. In order to avoid this reaction, not a polynucleotide but a low molecular weight compound is used as an internal standard substance.

  When such a standard is added to a sample (for example, blood) in advance and a polynucleotide is purified by a method using a commercially available column or by ethanol precipitation, there is an internal standard in the purified product. do not do. In other words, the internal standard is lost during the purification process. Therefore, in the conventional quantification method, an internal standard substance exhibiting fluorescence is added to the purified polynucleotide, and then the mixture is subjected to capillary electrophoresis.

  However, in such a conventional quantification method, it becomes impossible to determine the amount of the polynucleotide after correcting an error (for example, purification efficiency of the polynucleotide) generated in the process of purifying the polynucleotide. (In other words, only the error in injecting the sample into the capillary electrophoresis apparatus can be corrected). That is, the conventional quantification method has a problem in that erroneous data interpretation may be caused when the purification efficiency of the measurement sample and the purification efficiency of the calibration curve sample are different.

  The present invention has been made in view of the above-described conventional problems, and its purpose is low in cost, can be quantified in a short time, can quantitate various types of nucleic acids, and can be quantified. It is an object of the present invention to provide a highly accurate polynucleotide quantification method and electrophoresis method, and a method for preparing a sample used therefor.

  As a result of repeated studies to solve the above-mentioned problems, the present inventor quantified in a series of steps for quantifying a polynucleotide (for example, purification with an organic solvent, electrophoresis by electrophoresis, detection by laser-induced fluorescence detection). Using an internal standard that exhibits the same behavior as the target polynucleotide and subjecting both the polynucleotide and the internal standard to the series of steps described above, the measured amount of polynucleotide is used as the internal standard. It was found that if the correction was made based on the above, the problems of the conventional quantitative method described above could be solved, and the present invention was completed.

  In order to solve the above problems, a sample preparation method for use in electrophoresis of the present invention comprises mixing a sample solution containing a polynucleotide and a fluorescently labeled internal standard substance with an organic solvent, It includes a step of obtaining an aqueous phase.

  In the method for preparing a sample to be subjected to electrophoresis of the present invention, the fluorescently labeled internal standard substance is preferably transferred to the mixed aqueous phase in the step of obtaining the aqueous phase.

  In the method for preparing a sample for use in electrophoresis according to the present invention, the ratio of the mass of the fluorescently labeled internal standard substance transferred to the aqueous phase to the total mass of the fluorescently labeled internal standard substance is determined by the poly It is preferably substantially the same as the ratio of the mass of the polynucleotide transferred to the aqueous phase to the total mass of nucleotides.

  In the method for preparing a sample to be subjected to electrophoresis of the present invention, the ratio of the mass of the fluorescently labeled internal standard substance transferred to the aqueous phase to the total mass of the fluorescently labeled internal standard substance is represented by A (% ), And the ratio of the mass of the polynucleotide transferred to the aqueous phase to the total mass of the polynucleotide is B (%), the A (%) is 0.50 × B (%) to 1 .50 × B (%) is preferable.

  In the method for preparing a sample for use in electrophoresis of the present invention, the fluorescently labeled internal standard substance is fluorescein or an analog having a fluorescein skeleton (for example, Oregon Green (registered trademark) 488, Alexa Fluor (registered trademark)). 488, rhodamine), an analog having a BODIPY (registered trademark) or Dipyrromethene skeleton, a fluorescently labeled nucleotide, a fluorescently labeled deoxynucleotide, or a fluorescently labeled polynucleotide.

  In the method for preparing a sample for use in electrophoresis of the present invention, the fluorescently labeled internal standard substance is preferably a fluorescently labeled nucleotide or a fluorescently labeled deoxynucleotide.

  In the method for preparing a sample for use in electrophoresis of the present invention, the fluorescently labeled nucleotide and the fluorescently labeled deoxynucleotide are adenosine triphosphate, cytidine triphosphate, thymidine triphosphate, guanosine triphosphate. Acid, uridine triphosphate, adenosine diphosphate, cytidine diphosphate, thymidine diphosphate, guanosine diphosphate, uridine diphosphate, adenosine monophosphate, cytidine monophosphate, thymidine monophosphate, guanosine monophosphate It is preferable that the acid, uridine monophosphate, or an analog thereof is fluorescently labeled.

  In the method for preparing a sample to be subjected to electrophoresis of the present invention, the fluorescently labeled internal standard substance is preferably one that exhibits fluorescence at a wavelength of 470 nm to 700 nm.

  In the method for preparing a sample for use in electrophoresis of the present invention, the organic solvent is preferably an organic solvent containing phenol.

  In the method for preparing a sample to be subjected to electrophoresis of the present invention, the aqueous phase is preferably a sample to be subjected to electrophoresis.

  In order to solve the above problems, the electrophoresis method of the present invention is characterized by using a sample prepared by a method for preparing a sample for use in the electrophoresis of the present invention.

  The polynucleotide quantification method of the present invention is characterized by using the electrophoresis method of the present invention in order to solve the above problems.

  In order to solve the above-mentioned problems, the apparatus of the present invention is characterized by performing a separation analysis of a sample produced by the method of the present invention.

  The apparatus of the present invention is preferably an electrophoresis apparatus.

  The present invention quantifies polynucleotides using electrophoresis combined with laser-induced fluorescence detection. Therefore, according to the present invention, there is an effect that a small amount of polynucleotide can be quantified.

  In the conventional quantification method, it takes several hours to purify the polynucleotide. On the other hand, in the present invention, it is possible to carry out operations corresponding to these in several tens of minutes. Therefore, according to the present invention, there is an effect that the time required for quantification of the polynucleotide can be greatly reduced.

  The present invention does not require a column or the like for purifying the polynucleotide. Therefore, according to the present invention, there is an effect that the cost for quantifying the polynucleotide can be greatly reduced.

  In the conventional quantification method, it is necessary to select a method for purifying nucleotides according to the type of polynucleotide (DNA or RNA) to be quantified. On the other hand, in the present invention, quantification is performed using a purified product purified by a simple method, which can contain any kind of polynucleotide. Therefore, in this invention, there exists an effect that any kind of polynucleotide (DNA and RNA) can be quantified by the same method.

  In the present invention, after mixing a solution in which a polynucleotide to be quantified and an internal standard substance are mixed with an organic solvent to obtain an aqueous phase, a fluorescently labeled polynucleotide complementary to the quantified object is added to the aqueous phase. Nucleotides are added, and the solution containing the quantification target hybridized with the fluorescently labeled polynucleotide is subjected to electrophoresis. Then, the actually measured amount (concentration) of the polynucleotide is corrected based on the internal standard substance. In other words, the amount of polynucleotide after correction is not only an error in electrophoresis (for example, an error in the amount of injection that occurs when a sample is injected into an analyzer), but also purification of the polynucleotide (for example, using an organic solvent). The error in the purification efficiency that occurs during extraction) is also corrected. Therefore, according to the present invention, even if the purification efficiency of the measurement sample and the purification efficiency of the sample used for preparing the calibration curve are slightly different from each other, the amount of the polynucleotide can be accurately determined. Play.

It is a chromatogram which shows the detection result of the sample which hybridized DNA which consists of 21 bases, and the probe 1 by CE-LIF in the Example of this invention. It is a chromatogram which shows the detection result of the sample which hybridized microRNA which consists of 21 bases, and the probe 2 by CE-LIF in the Example of this invention.

  The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples are used. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention.

  In addition, when “A to B” is described in the present specification, the description intends “A or more and B or less”.

[1. Sample preparation method for electrophoresis]
A method for preparing a sample for use in electrophoresis according to the present embodiment (for example, electrophoresis combined with laser-induced fluorescence detection (for example, capillary electrophoresis combined with laser-induced fluorescence detection) is a polynucleotide (for example, , Oligonucleotide) and a fluorescently labeled internal standard substance and an organic solvent are mixed to obtain a mixed aqueous phase. If a sample solution and an organic solvent are mixed, the organic phase which has the said organic solvent as a main component, and an aqueous phase will isolate | separate. The aqueous phase contains both the polynucleotide and the internal standard substance, and the aqueous phase serves as a sample for use in electrophoresis. That is, the aqueous phase is subjected to electrophoresis without being subjected to the treatment of alcohol precipitation (for example, precipitation using alcohol such as ethanol or isopropanol).

  In other words, the method for preparing a sample for use in electrophoresis according to the present embodiment is a method for preparing a sample for use in electrophoresis without performing concentration treatment (for example, alcohol precipitation treatment) on the aqueous phase. Is the method.

  The specific form of the electrophoresis is not particularly limited, and may be, for example, capillary electrophoresis or electrophoresis using a plate-like gel.

  In the present specification, “nucleotide” means a nucleoside in which a phosphate group is ester-bonded. “Nucleoside” refers to a purine base or pyrimidine base bound to a sugar, which may be a modified purine base, a pyrimidine base may be a modified pyrimidine base, May be a modified sugar.

  As used herein, the term “polynucleotide” intends the same or different nucleosides linked through phosphodiester bonds. The portion of the phosphodiester bond may be thioated. Further, the “polynucleotide” in the present specification includes a polynucleotide constituted by DNA or RNA, and may be simply referred to as a nucleic acid. In addition, “polynucleotide” in the present specification means a polymer in which two or more nucleotides are linked, and the upper limit of the number of nucleotides constituting the polymer is not limited.

  As used herein, “oligonucleotide” refers to a polymer in which 2 or more and less than 100 nucleotides are linked, among polynucleotides.

  The sample solution only needs to contain at least a polynucleotide to be quantified and an internal standard, and the specific configuration is not particularly limited.

  For example, preparing a sample solution by adding a polynucleotide and an internal standard to a desired aqueous solution (eg, distilled water, physiological saline, phosphate buffer, Tris buffer, sodium acetate buffer, etc.) Can do.

  Alternatively, biological samples (eg, cells, tissues, urine, feces, blood, sputum, pus, microorganisms, viruses, culture fluids, etc.) or industrial products (eg, drinking water, food, pharmaceuticals, cosmetics) are liquefied. The sample solution can be prepared by adding an internal standard substance to the liquefied biological sample or industrial product. In this case, the polynucleotide contained in the biological sample or the industrial product becomes the object of quantification.

  The origin of the biological sample is not particularly limited, and may be a human, a non-human animal, or a vertebrate other than a human (eg, a pig, cow, sheep, chicken, etc.). It may be a microbe or a virus.

  A method for liquefying a biological sample or an industrial product is not particularly limited. For example, a desired aqueous solution (for example, distilled water, physiological saline, phosphate buffer, Tris buffer, acetic acid) is used for the biological sample or industrial product. Sodium buffer solution or the like) may be added, or a solid solution may be crushed with a mixer or the like after adding a desired aqueous solution to a biological sample or industrial product.

  The fluorescently labeled internal standard substance may be any substance that moves to the mixed aqueous phase in the step of obtaining the aqueous phase. According to the above configuration, since both the polynucleotide and the fluorescently labeled internal standard substance are present in the sample to be subjected to electrophoresis, the amount of the actually measured polynucleotide is accurately determined based on the internal standard substance. Can be corrected.

  More specifically, the total mass of the fluorescently labeled internal standard substance (in other words, the sum of the mass of the internal standard substance contained in the aqueous phase and the mass of the internal standard substance contained in the organic phase) The ratio of the mass of the fluorescently labeled internal standard substance transferred to the aqueous phase to the total mass of the polynucleotide (in other words, the mass of the polynucleotide contained in the aqueous phase and the mass contained in the organic phase) The ratio of the mass of the polynucleotide transferred to the aqueous phase relative to the total mass of the polynucleotide may be substantially the same. Of course, the two ratios may be the same.

  For example, let A (%) be the ratio of the mass of the fluorescently labeled internal standard substance transferred to the aqueous phase to the total mass of the fluorescently labeled internal standard substance, and transfer to the aqueous phase relative to the total mass of the polynucleotide. The mass ratio of the polynucleotide is B (%).

  In this case, the A (%) is preferably 0.50 × B (%) to 1.50 × B (%), and 0.60 × B (%) to 1.40 × B (%). More preferably, it is 0.70 × B (%) to 1.30 × B (%), more preferably 0.80 × B (%) to 1.20 × B (%). More preferably, 0.90 × B (%) to 1.10 × B (%) is preferable, and 0.91 × B (%) to 1.09 × B (%) is further preferable. Preferably, it is 0.92 × B (%) to 1.08 × B (%), more preferably 0.93 × B (%) to 1.07 × B (%), 0.94 × B (%) to 1.06 × B (%) is more preferable, 0.95 × B (%) to 1.05 × B (%) is further preferable, and 96 × B (%) ~ 1 04 × B (%) is more preferable, 0.97 × B (%) to 1.03 × B (%) is further preferable, and 0.98 × B (%) to 1.02 ×. B (%) is more preferable, 0.99 × B (%) to 1.01 × B (%) is further preferable, and 1.00 × B (%) is most preferable.

  In the step of obtaining the aqueous phase, the fluorescent-labeled internal standard substance is 10% by weight or more of the internal standard substance contained in the sample solution before being mixed, more preferably 20% by weight or more, more preferably 30% by weight or more, more preferably 40% by weight or more, more preferably 50% by weight or more, more preferably 60% by weight or more, more preferably 67% by weight or more, more preferably 70% by weight or more. More preferably, 80% by weight or more, more preferably 81% by weight or more, more preferably 90% by weight or more, more preferably 95% by weight or more, is transferred to the aqueous phase after mixing. preferable.

  The polynucleotide transitions to the aqueous phase, not the organic phase. Therefore, the polynucleotide can be quantified with higher accuracy as the internal standard substance shifts to the aqueous phase in the same manner as the polynucleotide, in other words, the higher the rate of transfer to the aqueous phase.

  The fluorescent-labeled internal standard substance has a fluorescein or an analog having a fluorescein skeleton (for example, Oregon Green (registered trademark) 488, Alexa Fluor (registered trademark) 488, rhodamine), BODIPY (registered trademark) or Dipyrrocene skeleton It may be an analog, a fluorescently labeled nucleotide, a fluorescently labeled deoxynucleotide, or a fluorescently labeled polynucleotide. Among these, it is preferable to use fluorescently labeled nucleotides or fluorescently labeled deoxynucleotides.

  The “fluorescein skeleton” means a skeleton in which a benzene ring is bonded to the 9-position of the 3H-xanthene skeleton. The “analog having a fluorescein skeleton” may be a compound in which an oxygen atom in the xanthene skeleton is substituted with a carbon atom, a silicon atom, a germanium atom, a tin atom, or a lead atom.

  In addition, the “analog having a Dipyrromethene skeleton” is intended to be a compound having a Dipyrromethene skeleton, and specific examples thereof include BODIPY FL (registered trademark) and BODIPY TMR (registered trademark).

  Since fluorescently labeled nucleotides and fluorescently labeled deoxynucleotides have a molecular structure similar to the polynucleotide to be quantified, a series of steps for quantifying the polynucleotide (for example, purification with an organic solvent, In migration by electrophoresis, detection by laser-induced fluorescence detection), the same behavior as the polynucleotide to be quantified is exhibited. Therefore, with the above configuration, the polynucleotide can be quantified with higher accuracy.

  Moreover, the fluorescence-labeled nucleotide and the fluorescence-labeled deoxynucleotide do not bind nonspecifically to the polynucleotide to be quantified and the probe that hybridizes to the polynucleotide. Therefore, with the above configuration, the polynucleotide can be quantified with higher accuracy.

  The specific configuration of the fluorescently labeled nucleotide is not particularly limited, but nucleotides (for example, adenosine triphosphate, cytidine triphosphate, thymidine triphosphate, guanosine triphosphate, uridine triphosphate, adenosine diphosphate) , Cytidine diphosphate, thymidine diphosphate, guanosine diphosphate, uridine diphosphate, adenosine monophosphate, cytidine monophosphate, thymidine monophosphate, guanosine monophosphate, uridine monophosphate, or these Analog) or deoxynucleotides fluorescently labeled.

Examples of the analog include the following. That means
(I) Nucleotides in which the phosphodiester linkage moiety is modified with thioate, dithioate, amidate, formacetal, 3′-amine, carbamate, or boranophosphate.

  (Ii) Modification of the sugar moiety in the nucleotide is not particularly limited. For example, a nucleotide in which the oxygen atom at the 2'-position, 3'-position, 4'-position and / or 5'-position is substituted with another atom.

For example, halogenation, O-alkylation (eg, O-methylation, O-ethylation, O-methoxyethylation, O-allylation), O-arylation, S-alkylation (eg, S-methylation) , S-ethylated, S-allylated), S-arylated or aminated (—NH ₂ ) nucleotides. “Aryl” refers to an aromatic group having at least one ring having a conjugated π-electron system, including carbocyclic aryl, heterocyclic aryl, and diaryl groups, all of which are optionally substituted. May be. Preferred substituents attached to the aryl group include halogen, trihalomethyl, hydroxyl, SH, cyano, alkoxy, alkyl, alkenyl, alkynyl and amino groups.

  (Iii) A nucleotide in which the oxygen atom at the 4 ′ position is substituted with sulfur, a nucleotide in which the 2 ′ position and the 4 ′ position are modified through methylene, and the 3 ′ position and the 4 ′ position are modified through methylene Nucleotides in which the hydroxyl group at the 3′-position or the 5′-position is substituted with an amino group, the hydroxyl group at the 5′-position is substituted with an amino group, and the 3′-position and the 5′-position are modified via methylene Nucleotides Nucleotides in which the sugar moiety is replaced from ribose to another sugar (eg glycerol, cyclohexene or throse).

  (Iv) Modification of the base in the nucleotide is not particularly limited. For example, nucleotides subjected to 5-position pyrimidine modification, 6-position, 7-position and / or 8-position purine modification (for example, O-methyl modification), exocyclic amines Modified nucleotides, nucleotides substituted with 4-thiouridine, 5-bromo modified nucleotides, 5-iodouracil modified nucleotides, 5-methylcytosine modified nucleotides.

  The method for fluorescently labeling the internal standard substance is not particularly limited. For example, a commercially available fluorescent dye (for example, fluorescein, BODIPY (registered trademark), Alexa 488 (registered trademark), etc.) is applied to the desired internal standard substance by a known method. What is necessary is just to connect.

  As the fluorescent dye, it is preferable to use the same fluorescent dye used for labeling the probe used for quantification of the polynucleotide. In other words, the fluorescently labeled internal standard substance and the fluorescently labeled probe used for quantification of the polynucleotide preferably exhibit fluorescence at the same wavelength.

  According to the above configuration, when the polynucleotide is quantified, the fluorescently labeled internal standard substance and the fluorescently labeled probe can be simultaneously detected and quantified with a simple apparatus.

  For example, the fluorescently labeled internal standard substance may exhibit fluorescence at a wavelength of 470 nm to 700 nm (for example, 520 nm, 560 nm, 655 nm, or 675 nm). Of course, the fluorescently labeled internal standard substance may exhibit fluorescence at wavelengths other than those described above.

  The sample solution may contain a protein denaturant (specifically, sodium dodecyl sulfate or protease). According to the above configuration, the activity of the protein that inhibits quantification can be reduced, or the protein that inhibits quantification can be removed, so that the polynucleotide can be quantified with higher accuracy.

  Although it does not specifically limit as a density | concentration of the protein denaturant in a sample solution, For example, 1.5% (w / v)-2% (w / v) may be sufficient. According to the above configuration, the polynucleotide can be quantified with higher accuracy without inhibiting the quantification.

  The sample solution may contain a hybridization buffer (50 mM Tris-Acetate (pH 8.0), 50 mM sodium chloride, 10 mM EDTA). According to this configuration, hybridization can be performed at an optimum salt concentration and pH.

  It does not specifically limit as an organic solvent used for the refinement | purification of a sample solution, For example, it may be an organic solvent containing a phenol. For example, as the organic solvent, “phenol”, “a mixture of phenol and chloroform”, “a mixture of phenol and isoamyl alcohol”, or “a mixture of phenol, chloroform and isoamyl alcohol” can be used. .

  The pH of the organic solvent is not particularly limited, but is preferably, for example, pH 7 to pH 8, and most preferably about pH 8.0. According to the said structure, both DNA and RNA can be transferred to an aqueous phase equally, As a result, both DNA and RNA can be quantified by the same method.

[2. Electrophoresis method
The electrophoresis method of the present embodiment is a method using a sample prepared by a method for preparing a sample for use in electrophoresis of the present invention.

  The specific form of electrophoresis is not particularly limited, and may be, for example, capillary electrophoresis or electrophoresis using a plate-like gel.

  The gel used for separation is not particularly limited, and a commercially available gel (for example, agarose gel, polyacrylamide gel, etc.) can be used as appropriate.

  Electrophoresis can be performed using a commercially available electrophoresis apparatus.

[3. (Polynucleotide quantification method)
The polynucleotide quantification method of the present embodiment may use the electrophoresis method of the present invention, and the specific configuration is not particularly limited.

  For example, the polynucleotide quantification method of the present embodiment can be configured by combining the electrophoresis method of the present invention and well-known laser-induced fluorescence detection.

  Hereinafter, an example of a method for quantifying a polynucleotide according to the present embodiment will be described, but the present invention is not limited to the following configuration.

  In the polynucleotide quantification method of the present embodiment, a fluorescently labeled probe that hybridizes to the polynucleotide is added to a sample to be subjected to electrophoresis prepared by the above-described method of the present invention to prepare a mixture. A mixing step, and subjecting the mixture to electrophoresis, and during the electrophoresis, a fluorescence signal derived from the fluorescently labeled internal standard substance, and a fluorescence signal derived from the probe hybridized to the polynucleotide; A ratio of a fluorescence signal area value derived from a probe hybridized to the polynucleotide to an area value of a fluorescence signal derived from the fluorescently labeled internal standard substance, and a previously prepared calibration The amount of polynucleotide contained in the sample solution is calculated based on the line It includes out and step.

  Below, each process is demonstrated. In addition, [1. The description of the configuration already described in the column “Method for preparing sample for electrophoresis” is omitted below.

[3-1. (Mixing process)
The mixing step is a step of preparing a mixture by adding a fluorescently labeled probe that hybridizes to a polynucleotide to a sample to be subjected to electrophoresis prepared by the method of the present invention. In the present invention, the polynucleotide and the probe are hybridized in the mixing step.

  The probe is not particularly limited as long as it hybridizes to the polynucleotide.

  For example, as the probe, DNA or RNA complementary to the polynucleotide to be quantified can be used. More specifically, DNA or RNA complementary to the full length or partial length of the polynucleotide to be quantified can be used as the probe.

  The method of fluorescently labeling the probe is not particularly limited. For example, if a commercially available fluorescent dye (for example, fluorescein, BODIPY (registered trademark), Alexa 488 (registered trademark)) is linked to a desired probe by a known method. Good.

  As the fluorescent dye, it is preferable to use the same fluorescent dye as that used for labeling the above-mentioned internal standard substance. In other words, the fluorescently labeled internal standard substance and the fluorescently labeled probe preferably exhibit fluorescence at the same wavelength.

  According to the above configuration, when the polynucleotide is quantified, the fluorescently labeled internal standard substance and the fluorescently labeled probe can be simultaneously detected and quantified with a simple apparatus.

  For example, the fluorescently labeled probe may exhibit fluorescence at a wavelength of 470 nm to 700 nm (eg, 520 nm, 560 nm, 655 nm, or 675 nm). Of course, the fluorescently labeled probe may exhibit fluorescence at wavelengths other than those described above.

  Although the temperature of the mixture in the said mixing process is not specifically limited, It is also possible to set to the temperature which can inhibit nonspecific hybridization. In this case, the temperature may be set according to the base sequence of the probe. Specifically, it can be set based on the number of bases of the probe, the GC content of the probe, the Tm value of the probe, and the like.

  For example, it can be set to 44 ° C. to 48 ° C., and can be set to 52 ° C. to 60 ° C. Of course, the present invention is not limited to the temperature.

[3-2. Detection process)
In the detection step, the mixture described above is subjected to electrophoresis, and during the electrophoresis, a fluorescent signal derived from a fluorescently labeled internal standard substance and a fluorescent signal derived from a probe hybridized to a polynucleotide are detected. It is a process to do.

  Specific methods of electrophoresis and laser-induced fluorescence detection are not particularly limited, and may be appropriately performed using a commercially available apparatus and a protocol attached to the apparatus.

[3-3. Calculation process]
The calculation step is based on the ratio of the area value of the fluorescence signal derived from the probe hybridized to the polynucleotide to the area value of the fluorescence signal derived from the fluorescently labeled internal standard substance, and the calibration curve prepared in advance. This is a step of calculating the amount of polynucleotide contained in the sample solution which is the first sample.

  That is, in the calculation step, first, the ratio of the amount of polynucleotide finally detected to the amount of internal standard substance finally detected is calculated. Then, the ratio is a function of a calibration curve prepared in advance (for example, the concentration (known concentration) of the polynucleotide contained in the sample solution, which is the initial sample, and the ratio (of the fluorescence signal actually measured). By substituting it into a function indicating the relationship with the ratio calculated from the area value, the amount of polynucleotide contained in the sample solution, which is the first sample, is calculated.

  At this time, since the amount of polynucleotide contained in the sample solution, which is the initial sample, is calculated using the above-described ratio and calibration curve, a series of steps for quantifying the polynucleotide (for example, depending on the organic solvent) The amount of polynucleotide contained in the sample solution, which is the first sample, is calculated in a state where various errors generated in purification, electrophoresis by electrophoresis, and detection by laser-induced fluorescence detection are corrected. As a result, the polynucleotide can be quantified with higher accuracy.

  The fluorescence signal can be shown as a chromatogram (in other words, a peak in the chromatogram) in which the fluorescence intensity at the time is plotted with respect to the fluorescence measurement time (see, for example, FIGS. 1 and 2). The “area value of the fluorescence signal” is intended to be the area of the region surrounded by the peak and the fluorescence intensity baseline.

  Although the specific configuration of the calibration curve is not particularly limited, for example, a fluorescently labeled internal standard obtained using a sample solution containing a known concentration of polynucleotide and a known concentration of fluorescently labeled internal standard substance It may be a function of the ratio of the area value of the fluorescent signal derived from the probe hybridized to the polynucleotide to the area value of the fluorescent signal derived from the substance and the known concentration of the polynucleotide. Below, the preparation method of the said calibration curve is further demonstrated.

  First, a plurality of sample solutions having various concentrations and a known concentration of polynucleotide and a fixed concentration of fluorescently labeled internal standard substance are prepared.

  Next, the plurality of sample solutions are subjected to the mixing step and the detection step described above, and for each sample solution, a fluorescent signal derived from a fluorescently labeled internal standard substance and a fluorescent signal derived from a probe hybridized to the polynucleotide , Is detected. Then, the ratio of the area value of the fluorescence signal derived from the probe hybridized to the polynucleotide to the area value of the fluorescence signal derived from the fluorescently labeled internal standard substance is calculated.

  From the above, for each sample solution that is the initial sample, the correspondence between the known concentration of the polynucleotide in the sample solution and the actually measured ratio is clarified.

  The data of each sample solution is then plotted on the XY plane with the known concentration of the polynucleotide as the X axis and the ratio as the Y axis.

  Furthermore, a function (for example, Y = aX + b, a and b are constants determined by experiments) that approximate the above data is specified by a well-known method (for example, least square method), and the function is used as a calibration curve. be able to.

  In actual quantification, since the ratio (value of Y) is determined based on the actually measured data in the calculation step, a series of sequences for quantifying polynucleotides can be obtained by substituting the ratio into the function described above. The amount (concentration) of polynucleotide in the sample solution, which is the first sample, with various errors corrected in the process (for example, purification by organic solvent, electrophoresis by electrophoresis, detection by laser-induced fluorescence detection). Will be calculated.

  Although an example of a calibration curve has been described above, the present invention is of course not limited to the calibration curve, and a desired calibration curve can be used as appropriate.

  The polynucleotide quantification method of the present embodiment can be configured as follows.

  The polynucleotide quantification method of the embodiment includes a mixing step of preparing a mixture by adding a fluorescently labeled probe that hybridizes to the polynucleotide to a sample to be subjected to electrophoresis prepared by the method of the present invention. And the mixture is subjected to electrophoresis, and during the electrophoresis, a fluorescent signal derived from the fluorescently labeled internal standard substance and a fluorescent signal derived from the probe hybridized to the polynucleotide are detected. The ratio of the area value of the fluorescent signal derived from the probe hybridized to the polynucleotide to the area value of the fluorescent signal derived from the fluorescently labeled internal standard substance and the calibration curve prepared in advance. Based on the calculation step of calculating the amount of the polynucleotide contained in the sample solution, It may include.

  The calibration curve is obtained by using the polynucleotide for the area value of the fluorescence signal derived from the fluorescently labeled internal standard substance obtained using a sample solution containing the polynucleotide and the fluorescently labeled internal standard substance at a known concentration. It may be a function of the ratio of the area values of fluorescent signals derived from probes hybridized to nucleotides and the known concentration.

  The fluorescently labeled probe may exhibit fluorescence at a wavelength of 470 nm to 700 nm (for example, 520 nm, 560 nm, 655 nm, or 675 nm).

[4. apparatus〕
The apparatus of this embodiment is characterized in that the sample prepared by the method for preparing a sample for use in electrophoresis of the present invention is separated and analyzed. In other words, the apparatus of this embodiment is characterized in that the sample is separated and analyzed using the method for preparing a sample for use in electrophoresis of the present invention.

  According to the above configuration, a small amount of polynucleotide can be separated and analyzed in a short time, at low cost, and with high accuracy. Moreover, according to the said structure, each of DNA and RNA can be separated and analyzed using the apparatus of the same structure.

  The apparatus of the present embodiment can be an electrophoresis apparatus, for example.

  The specific configuration of the apparatus of the present embodiment is not particularly limited. For example, the configuration for carrying out the sample preparation method for use in the electrophoresis of the present invention, in other words, the sample for use in the electrophoresis of the present invention. A sample preparation unit for preparing a sample may be provided according to the sample preparation method.

  Further, the apparatus of the present embodiment does not include the sample preparation section, but includes a sample introduction section for introducing a sample prepared according to the sample preparation method for use in electrophoresis of the present invention into the apparatus. You may have.

  The sample preparation unit includes, for example, a mixing unit for mixing a sample solution containing a polynucleotide and a fluorescently labeled internal standard substance and an organic solvent, and a recovery unit for recovering the mixed aqueous phase. obtain.

  The mixing unit may include, for example, a container for storing the sample solution and the organic solvent, and a blade for mixing the sample solution and the organic solvent in the container.

  The said container and a blade are not specifically limited, A well-known container and a blade can be used suitably. For example, the container and the blade are preferably formed of a material that does not deteriorate with an organic solvent.

  The mixing unit includes, for example, a container for storing the sample solution and the organic solvent, and a vibrating unit for vibrating the container (for example, movement in the vertical direction with respect to the container, horizontal direction And / or a vibrating part that generates a rotational motion).

  The specific configuration of the container and the vibration part is not particularly limited, and a well-known container and vibration part can be used. For example, the container is preferably formed of a material that does not deteriorate with an organic solvent.

  The recovery unit is not particularly limited as long as it can recover the aqueous phase. For example, the collection unit may be various syringes. If the solution mixed in the mixing part is allowed to stand for a predetermined time, it is separated into an aqueous phase and an organic phase. Then, it is only necessary to collect only the aqueous phase with various syringes. In this case, the aqueous phase collected by various syringes may be provided to an electrophoresis gel disposed in the apparatus.

  The sample introduction part may be any one that can introduce a sample prepared according to a sample preparation method for use in electrophoresis of the present invention into the apparatus.

  For example, the sample introduction unit may include an opening provided on the surface of the device, and a storage unit (in other words, a space) for storing a sample introduced into the device from the opening.

  In this case, the storage unit may be connected to an electrophoresis gel disposed in the apparatus, and the sample stored in the storage unit may be provided to the electrophoresis gel.

  The apparatus according to the present embodiment may include a detection unit for detecting fluorescence having a wavelength of 470 nm to 700 nm.

  The specific configuration of the detection unit is not particularly limited, and a known detection device can be appropriately used as the detection unit.

  In addition to the above-described configuration, the device according to the present embodiment can include various configurations included in a known device or a known electrophoresis device.

[Example 1] Purification of DNA 20 μL of an aqueous solution containing SDS (sodium dodecyl sulfate) at a concentration of 20% (w / v) is added to 160 μL of human plasma, so that components contained in human plasma are solubilized. An aqueous solution was prepared.

21-base DNA (Integrated DNA Technologies, Inc) is dissolved in a TE solution (pH 8.0 (Nacalai Tesque)), and the aqueous solution is further diluted with a hybridization buffer to contain 21-base DNA. An aqueous solution was prepared. The base sequence of the DNA is as follows. That means
DNA: 5'-GCGTTTGCCTTCTCTTCTTGCG-3 '... (SEQ ID NO: 1).

  10 μL of an aqueous solution containing DNA consisting of 21 bases is added to an aqueous solution in which components contained in human plasma are solubilized, and 10 μL of an aqueous solution containing fluorescein, an internal standard substance, at a concentration of 500 nM is added. A mixed solution was prepared.

  200 μL of a mixture of organic solvents (phenol: chloroform: isoamyl alcohol = 25: 24: 1) (Sigma-Aldrich Co. LLC.) Saturated with TE solution (pH 8.0) was added to the above mixed solution. After stirring and mixing, the mixture was centrifuged at 10,000 rpm for 5 minutes at room temperature.

  90 μL of the supernatant separated by centrifugation was collected and used as a sample solution containing DNA.

[Example 2] Purification of microRNA 20 μL of an aqueous solution containing SDS at a concentration of 20% (w / v) was added to 160 μL of human plasma to prepare an aqueous solution in which components contained in human plasma were solubilized. .

A 21-base microRNA (Integrated DNA Technologies, Inc) is dissolved in a TE solution (pH 8.0 (Nacalai Tesque)), and the aqueous solution is further diluted with a hybridization buffer to contain a 21-base microRNA. An aqueous solution was prepared. The base sequence of the microRNA is as follows. That means
microRNA: 5'-UUAAGACUUGCAGUGAUGUUU-3 '... (SEQ ID NO: 2).

  10 μL of an aqueous solution containing microRNA consisting of 21 bases is added to an aqueous solution in which components contained in human plasma are solubilized, and BODIPY (registered trademark) FL ATP (Life Technologies Corporation.) Which is an internal standard substance is added 10 μL of an aqueous solution containing a concentration of 500 nM was added to prepare a mixed solution.

  200 μL of a mixture of organic solvents (phenol: chloroform: isoamyl alcohol = 25: 24: 1) (Sigma-Aldrich Co. LLC.) Saturated with TE solution (pH 8.0) was added to the above mixed solution. After stirring and mixing, the mixture was centrifuged at 10,000 rpm for 5 minutes at room temperature.

  90 μL of the supernatant separated by centrifugation was collected and used as a sample solution containing microRNA.

[Example 3] Hybridization reaction between probe and DNA or microRNA 3'-FAM labeled oligonucleotide complementary to DNA consisting of 21 bases described in Example 1 (hereinafter referred to as oligonucleotide 1), Also, a 3′-FAM labeled oligonucleotide (hereinafter referred to as oligonucleotide 2) complementary to the microRNA consisting of 21 bases described in Example 2 was purchased from Integrated DNA Technologies, Inc (FAM: Ex / Em). = 488 nm / 520 nm). In addition, the base sequence of each oligonucleotide is as follows. That means
Oligonucleotide 1: 5′-CGCAAGAAGAGAGCAAACGC-3 ′ (SEQ ID NO: 3);
Oligonucleotide 2: 5'-AAACACTACTGCAAGTCTTAA-3 '... (SEQ ID NO: 4).

  To the 90 μL sample solution prepared in Example 1, 10 μL of the probe 1 (the amount of the probe was 50 nM) was added to make the total amount 100 μL. Then, the mixture was incubated at an optimal temperature for 30 minutes to hybridize DNA consisting of 21 bases and probe 1.

  In addition, 10 μL of the probe 2 (the amount of the probe was 50 nM) was added to the 90 μL sample solution prepared in Example 2 to make the total amount 100 μL. Then, the mixture was incubated at an optimal temperature for 30 minutes, and the microRNA consisting of 21 bases and the probe 2 were hybridized.

  The sample hybridized as described above was used in Examples described later.

[Example 4] Detection by CE-LIF (Capillary Electrophoresis-Laser Induced Fluorescence) The hybridized sample prepared in Example 3 was subjected to capillary electrophoresis under the following conditions and detected by a laser-induced fluorescence detection method. did.
Capillary electrophoresis apparatus: PA 800 plus (Beckman Coulter, Inc.),
Capillary: 20.0 cm (effective length), 30.2 cm (full length), 75 μm (inner diameter) (manufactured by GL Science Co., Ltd.)
-Capillary temperature: 30 ° C
-Running buffer: 25 mM sodium tetraborate (pH 9.2),
・ Detection: Laser-induced fluorescence detection,
Separation conditions (in the case of a sample in which DNA consisting of 21 bases and probe 1 are hybridized): 21 kV from 0 to 10 minutes,
Separation conditions (in the case of a sample in which microRNA consisting of 21 bases and probe 2 are hybridized): 12 kV from 0 to 10 minutes, and 12 kV from 10 to 12 minutes.

  FIG. 1 shows the detection result of a sample obtained by hybridizing DNA consisting of 21 bases and probe 1, and FIG. 2 shows the detection result of a sample obtained by hybridizing microRNA consisting of 21 bases and probe 2.

  Specifically, in FIG. 1, “IS” indicates fluorescein which is an internal standard substance, “unreacted probe” indicates probe 1 that does not hybridize to DNA consisting of 21 bases, and “DNA-DNA hybrid” "Indicates the probe 1 hybridized with DNA consisting of 21 bases.

  On the other hand, in FIG. 2, “IS” indicates BODIPY (registered trademark) FL ATP, which is an internal standard substance, and “unreacted probe” indicates probe 2 that does not hybridize to microRNA consisting of 21 bases. “miRNA-DNA hybrid” indicates a probe 2 hybridized with a 21-base microRNA.

  From FIG. 1, fluorescein, which is an internal standard substance, and DNA consisting of 21 bases are equivalent to an aqueous phase (in other words, when treated with a mixture of organic solvents saturated with TE solution (pH 8.0)). It became clear that the above-mentioned “sample solution containing DNA”) was transferred.

  In addition, although some organic solvent (for example, phenol) remains in the above-mentioned “sample solution containing DNA”, even if such an organic solvent is present, the high concentration of DNA consisting of 21 bases and probe 1 is high. It became clear that there was no effect on hybridization.

  On the other hand, from FIG. 2, BODIPY (registered trademark) FL ATP, which is an internal standard substance, and microRNA consisting of 21 bases are equivalent when treated with a mixture of organic solvents saturated with TE solution (pH 8.0). It was clarified that the phase shifts to the aqueous phase (in other words, the above-mentioned “sample solution containing microRNA”).

  Further, some organic solvent (for example, phenol) remains in the above-mentioned “sample solution containing microRNA”. Even if such an organic solvent is present, the high concentration of the microRNA consisting of 21 bases and the probe 2 is high. It became clear that there was no effect on hybridization.

[Example 5] Confirmation of linearity of detection data by CE-LIF A DNA consisting of 21 bases was dissolved in a TE solution (pH 8.0 (Nacalai Tesque)) and further diluted with a hybridization buffer to obtain 21 bases. Aqueous solutions containing various concentrations of DNA were prepared.

  Specifically, a plurality of mixed solutions containing DNA at 0 pM, 50 pM, 100 pM, 250 pM, 500 pM, 1000 pM, 2000 pM, 3000 pM, 4000 pM, or 5000 pM were prepared.

  In addition, microRNA consisting of 21 bases is dissolved in TE solution (pH 8.0 (Nacalai Tesque)) and further diluted with a hybridization buffer to prepare aqueous solutions containing microRNAs consisting of 21 bases at various concentrations. did.

  Specifically, a plurality of mixed solutions containing microRNA at 0 pM, 50 pM, 100 pM, 1000 pM, 2000 pM, 3000 pM, 4000 pM, or 5000 pM were prepared.

  The aqueous solutions having the respective concentrations described above were treated by the methods of Examples 1 to 3, and subjected to capillary electrophoresis under the conditions described in Example 4 and detected by a laser-induced fluorescence detection method.

  As described above, in this embodiment, a capillary electrophoresis apparatus manufactured by Beckman Coulter, Inc. is used to perform laser-induced fluorescence detection. In this example, the amount of the polynucleotide (in other words, the concentration) is calculated from the area value of the detected fluorescence signal by a program incorporated in the capillary electrophoresis apparatus.

  In brief, the concentration of DNA in the mixed solution is calculated from the area value of the fluorescence signal indicated by the probe 1 hybridized to the DNA consisting of 21 bases, and the fluorescence indicated by the probe 2 hybridized to the microRNA consisting of 21 bases. The concentration of microRNA in the mixed solution was calculated from the area value of the signal.

  Then, the linearity of the calculated data was confirmed by comparing the concentration of DNA or microRNA actually contained in the mixed solution with the calculated concentration.

  Table 1 shows test data on a mixed solution containing DNA, and Table 2 shows test data on a mixed solution containing microRNA. In Tables 1 and 2, “Nominal concentration” indicates the theoretical concentration of DNA or microRNA actually contained in the mixed solution used in the test. “Determined concentration” indicates the concentration of DNA or microRNA contained in the mixed solution calculated from the intensity of the detected fluorescence and the time when the fluorescence was detected. “Accuracy” indicates a difference between “Nominal concentration” and “Determined concentration”. “S1” to “S9” are names given to the mixed solutions.

  As shown in Table 1 and Table 2, the concentration of DNA or microRNA actually contained in the mixed solution is very close to the calculated concentration, and the calculated data has linearity. It was confirmed that

[Example 6] Addition and recovery of polynucleotide in biological sample To 160 μL of human plasma, 20 μL of an aqueous solution containing SDS (sodium dodecyl sulfate) at a concentration of 20% (w / v) is added to human plasma. An aqueous solution in which the contained components were solubilized was prepared.

  DNA consisting of 21 bases was dissolved in TE solution (pH 8.0 (Nacalai Tesque)) to prepare aqueous solutions containing DNA consisting of 21 bases at various concentrations. Moreover, microRNA consisting of 21 bases was dissolved in a TE solution (pH 8.0 (Nacalai Tesque)) to prepare aqueous solutions containing microRNA consisting of 21 bases at various concentrations. Each aqueous solution was further diluted with a hybridization buffer to prepare an aqueous solution containing DNA consisting of 21 bases or an aqueous solution containing microRNA consisting of 21 bases.

  10 μL of an aqueous solution containing DNA consisting of 21 bases is added to an aqueous solution in which components contained in human plasma are solubilized, and 10 μL of an aqueous solution containing fluorescein, an internal standard substance, at a concentration of 500 nM is added. A plurality of mixed solutions were prepared (n = 5). The addition concentration of DNA in each mixed solution was 50 pM, 150 pM, 2500 pM, 4000 pM, and 5000 pM.

  In addition, 10 μL of an aqueous solution containing microRNA consisting of 21 bases is added to an aqueous solution in which components contained in human plasma are solubilized, and BODIPY (registered trademark) FL ATP (Life Technologies Corporation. Was added at a concentration of 500 nM to prepare a plurality of mixed solutions (n = 5). The addition concentration of microRNA in each mixed solution was 50 pM, 150 pM, 2500 pM, 4000 pM, and 5000 pM.

  For each of the above mixed solutions, 200 μL of a mixture of organic solvents (phenol: chloroform: isoamyl alcohol = 25: 24: 1) (Sigma-Aldrich Co. LLC.) Saturated with TE solution (pH 8.0) After addition, stirring and mixing, the mixture was centrifuged at 10,000 rpm for 5 minutes at room temperature.

  90 μL of the supernatant separated by centrifugation was collected and used as a sample solution containing DNA or microRNA.

  To each 90 μL sample solution containing DNA, 10 μL of probe 1 (the amount of the probe was 50 nM) was added to make a total volume of 100 μL. Then, the mixture was incubated at an optimal temperature for 30 minutes to hybridize DNA consisting of 21 bases and probe 1.

  Further, 10 μL of the probe 2 (the amount of the probe was 50 nM) was added to each 90 μL of the sample solution containing microRNA to make the total amount 100 μL. Then, the mixture was incubated at an optimal temperature for 30 minutes, and the microRNA consisting of 21 bases and the probe 2 were hybridized.

  The hybridized sample was subjected to capillary electrophoresis under the same conditions as in Example 4 and detected by a laser-induced fluorescence detection method. The calibration curve of Example 5 was used to calculate the concentration in the sample.

  Table 3 shows test data relating to a mixed solution containing DNA, and Table 4 shows test data of a mixed solution containing microRNA. In Tables 3 and 4, “Nominal concentration” indicates the theoretical concentration of DNA or microRNA actually contained in the mixed solution used in the test. “Average Determined concentration” indicates the average concentration of DNA or microRNA contained in the mixed solution, which is calculated from the intensity of detected fluorescence and the time at which fluorescence is detected, in n = 5 measurement. “Accuracy” indicates a difference between “Nominal concentration” and “Determined concentration”. “Precision” indicates a variation in n = 5 measurement. Further, “LLOQ”, “L”, “M”, “H”, and “ULOQ” are names given to the respective mixed solutions.

  As shown in Table 3 and Table 4, even when a mixed solution containing various impurities derived from plasma was used, the concentration of DNA or microRNA actually contained in the mixed solution was calculated. The concentration was very close, and the calculated data confirmed that the addition was good.

[Example 7] Comparison of purification efficiency of DNA or microRNA and internal standard substance in purification using a mixture of organic solvents In Example 1 and Example 2, a mixed solution was purified using a mixture of organic solvents. ing. Therefore, in this Example 7, the purification efficiency of the internal standard substance in the purification was compared with the purification efficiency of the polynucleotide. The purification of the polynucleotide was confirmed using the probe 1 described above.

  40 μL of an aqueous solution containing SDS (sodium dodecyl sulfate) at a concentration of 20% (w / v) was added to 320 μL of ultrapure water, and 20 μL of a solution containing the probe 1 at a concentration of 20 nM, and 20 nM 20 μL of a solution containing an internal standard substance (fluorescein, BODIPY FL ATP, BODIPY FL GTP, BODIPY FL GDP, Oregon Green, Rhodamine Green, Alexa Fluor 488-dUTP, Alexa Fluor 488) at a concentration.

  Next, 200 μL of the above solution was collected, and a mixture of organic solvents (phenol: chloroform: isoamyl alcohol = 25: 24: 1) (Sigma-Aldrich Co. LLC.) Saturated with TE solution (pH 8.0) was added. After adding 200 μL, stirring and mixing, the mixture was centrifuged at 10000 rpm for 5 minutes at room temperature.

  100 μL of the supernatant separated by centrifugation was collected.

  Each of the aqueous solution before purification with an organic solvent and the supernatant obtained after purification were subjected to capillary electrophoresis under the same conditions as in Example 4 and detected by laser-induced fluorescence detection.

  The area value of the probe 1 relative to the area value of the internal standard substance was calculated from the detected area value of the internal standard substance and the fluorescence of the probe 1 (before the organic solvent mixture treatment: Ratio1, after the organic solvent mixture treatment: Ratio2) .

  Furthermore, by calculating the ratio of Ratio1 to Ratio2, the difference in the transition of the polynucleotide and the internal standard substance to the aqueous phase was confirmed. That is, when the ratio is greater than 1, the internal standard substance has a higher transferability to the aqueous phase than the polynucleotide, and when the ratio is 1, the transferability to the aqueous phase is equivalent. When is smaller than 1, it indicates that the internal standard substance has a lower ability to migrate to the aqueous phase than the polynucleotide. Table 5 shows the test data.

  As shown in Table 5, when purified with an organic solvent, it was revealed that the internal standard substance has the ability to migrate to an aqueous phase equivalent to a polynucleotide.

  The present invention can be used for quantification of polynucleotides contained in various samples (for example, biological samples (for example, blood, plasma, urine, cell debris), chemical samples).

  The invention relates to the preparation of a sample for electrophoresis (more specifically, electrophoresis combined with laser-induced fluorescence detection (more specifically, capillary electrophoresis combined with laser-induced fluorescence detection), and , And can be used in a method for quantifying polynucleotides using the sample.

Claims (10)

A method for preparing a sample for use in electrophoresis, comprising a step of mixing a sample solution containing a polynucleotide and a fluorescently labeled internal standard substance and an organic solvent to obtain an aqueous phase after mixing. The fluorescently labeled internal standard substance moves to the mixed aqueous phase in the step of obtaining the aqueous phase, and the aqueous phase with respect to the total mass of the fluorescently labeled internal standard substance The ratio of the mass of the fluorescently labeled internal standard substance that migrates to is substantially the same as the ratio of the mass of the polynucleotide that migrates to the aqueous phase with respect to the total mass of the polynucleotide. . The ratio of the mass of the fluorescent-labeled internal standard substance transferred to the aqueous phase to the total mass of the fluorescent-labeled internal standard substance is A (%), and the total phase of the polynucleotide is based on the total mass of the polynucleotide. When the mass ratio of the polynucleotide to be transferred is B (%), the A (%) is 0.50 × B (%) to 1.50 × B (%). A method for preparing a sample for use in electrophoresis according to Item 1 . The fluorescently labeled internal standard substance is fluorescein or an analog having a fluorescein skeleton, an analog having a BODIPY (registered trademark) or Dipyrrothene skeleton, a fluorescently labeled nucleotide, a fluorescently labeled deoxynucleotide, or a fluorescently labeled A method for preparing a sample for use in electrophoresis according to claim 1 or 2 , wherein the sample is a polynucleotide. The method for preparing a sample for use in electrophoresis according to claim 3 , wherein the fluorescently labeled internal standard substance is a fluorescently labeled nucleotide or a fluorescently labeled deoxynucleotide. The fluorescently labeled nucleotide and the fluorescently labeled deoxynucleotide are adenosine triphosphate, cytidine triphosphate, thymidine triphosphate, guanosine triphosphate, uridine triphosphate, adenosine diphosphate, cytidine diphosphate. Acid, thymidine diphosphate, guanosine diphosphate, uridine diphosphate, adenosine monophosphate, cytidine monophosphate, thymidine monophosphate, guanosine monophosphate, uridine monophosphate, or their analogs are fluorescent The method for preparing a sample for use in electrophoresis according to claim 3 or 4 , wherein the sample is labeled. The preparation of a sample for use in electrophoresis according to any one of claims 1 to 5 , wherein the fluorescently labeled internal standard substance exhibits fluorescence at a wavelength of 470 nm to 700 nm. Method. The method for preparing a sample for use in electrophoresis according to any one of claims 1 to 6 , wherein the organic solvent is an organic solvent containing phenol. The method for preparing a sample for use in electrophoresis according to any one of claims 1 to 7 , wherein the aqueous phase is a sample subjected to electrophoresis. An electrophoresis method using the sample prepared by the method according to any one of claims 1 to 8 . A method for quantifying a polynucleotide, wherein the electrophoresis method according to claim 9 is used.

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