JP2012223167A - Method for electrophoresing nucleic acid, method for concentrating and purifying nucleic acid, cartridge for nucleic acid electrophoresis, and method for producing the cartridge - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for electrophoresing nucleic acids, which is capable of highly efficiently concentrating and purifying nucleic acids in a short time by simple operation.SOLUTION: The method for electrophoresing nucleic acids includes a step wherein nucleic acids, into each of which an intercalator having an anionic functional group is intercalated, are electrophoresed. An acid dissociation constant of the intercalator can be 0 or more and 3 or less. The functional group can be a sulfo group. In the method for concentrating and recovering nucleic acids, the speed of nucleic acid electrophoresis can be increased by increasing the negative charges of the nucleic acids, each of which is bound to an intercalator.

Description

  The present technology relates to a nucleic acid electrophoresis method, a nucleic acid concentration purification method, a cartridge for nucleic acid electrophoresis, and a method for producing the cartridge. More specifically, the present invention relates to a cartridge for concentrating and purifying nucleic acid by electrophoresis in a channel.

  Nucleic acid amplification reactions such as PCR (Polymerase Chain Reaction) and LAMP (Loop-Mediated Isothermal Amplification) have been applied in various fields in biotechnology. For example, in the medical field, diagnosis based on the base sequence of DNA or RNA is performed, and in the agricultural field, DNA identification is used for determination of genetically modified crops.

  In the nucleic acid amplification reaction, nucleic acid in a small amount of sample can be amplified and detected with high efficiency. However, when the amount of nucleic acid contained in the sample is extremely small, it may be less than the lower limit of detection. Furthermore, when the nucleic acid concentration in the sample is extremely low, detection may not be possible because the nucleic acid to be amplified is not contained in a volume of the sample that can be introduced into the reaction field. In these cases, it is effective to concentrate the nucleic acid in the sample in advance and introduce it into the reaction field.

  Conventionally, as a nucleic acid concentration method, a method using phenol / chloroform / ethanol, a method using a column or filter for adsorbing nucleic acid, a method using magnetic silica beads, and the like are known. For example, Patent Document 1 discloses a nucleic acid concentration method using a porous carrier having nucleic acid adsorption ability. Non-Patent Document 1 describes a method of concentrating nucleic acids by capillary electrophoresis.

JP-A-2005-080555

"On-line sample preconcentration in capillary electrophoresis: Fundamentals and applications." Journal of Chromatography A., Vol.1184 (2008) p.504-541

  The conventional method using phenol / chloroform / ethanol requires the use of a harmful organic solvent, which takes time and effort for the centrifugation operation. In addition, the method using a column or filter that adsorbs nucleic acid is liable to cause clogging of the column or filter, and there is a problem from the simplicity of operation.

  Therefore, the main object of the present technology is to provide a nucleic acid electrophoresis method that is simple in operation and can concentrate and purify nucleic acids with high efficiency in a short time.

In order to solve the above problems, the present technology provides a nucleic acid electrophoresis method including a procedure for electrophoresis of a nucleic acid into which an intercalator having an anionic functional group is inserted.
In the procedure of this nucleic acid electrophoresis method, the acid dissociation constant of the intercalator is preferably greater than 0 and 3 or less.
Furthermore, in this nucleic acid electrophoresis method, the functional group is preferably a sulfo group.
Further, in this nucleic acid electrophoresis method, a voltage is applied to both ends of a channel filled with a buffer, the nucleic acid introduced into a predetermined location in the channel is electrophoresed, and the nucleic acid moving to the positive end is blocked. Preferably it includes a procedure.
Here, inserting an intercalator into a nucleic acid mainly means inserting an intercalator between complementary base pairs of a double-stranded nucleic acid. In the present technology, the double-stranded nucleic acid includes a nucleic acid that is originally composed of a single strand but is partially double-stranded by self-hybridization.
The pH of the buffer solution is preferably 5 or less. The pH of the buffer solution is preferably 4 or less. Furthermore, the pH of the buffer solution is preferably 3 or less.
The present technology also provides a method for concentrating and purifying nucleic acid, including a procedure for electrophoresis of a nucleic acid into which an intercalator having an anionic functional group is inserted.

  In addition, in the present technology, a channel into which a liquid can be introduced is formed, a blocking portion for blocking a substance is disposed at a predetermined position of the channel, electrodes are disposed at both ends of the channel, and an anionic functional group is provided. Provided is a cartridge for nucleic acid electrophoresis in which an intercalator that can be inserted into a nucleic acid and a buffer solution are accommodated between a damming portion and a negative electrode end.

  Furthermore, the present technology provides a step of forming a polymer gel or arranging a dialysis membrane by gelling a monomer solution introduced into a channel formed on a substrate into a predetermined shape by photopolymerization. And a step of accommodating an intercalator having an anionic functional group in a channel and capable of being inserted into a nucleic acid, and a method for producing a cartridge for nucleic acid electrophoresis.

  The present technology provides a nucleic acid electrophoresis method that is simple to operate and can concentrate and recover nucleic acids in a short time with high efficiency.

It is a schematic diagram explaining the structure of the cartridge for nucleic acid electrophoresis which concerns on each embodiment of this technique, and the procedure of the nucleic acid electrophoresis method using this cartridge. It is a pH-charge curve for demonstrating the acid dissociation constant which concerns on 1st Embodiment of this technique. It is a graph which shows a time-dependent change of the fluorescence intensity of the gel interface after the electrophoresis start (Example 1: pH of a buffer solution; 2.2). It is a graph which shows a time-dependent change of the fluorescence intensity of the gel interface after the electrophoresis start (Example 2: pH of a buffer solution; 4.0). It is a figure which shows the state which electrophoresed protein (BSA) (Example 3). It is a figure which shows the state which electrophoresed protein (AGP) (Example 4).

Hereinafter, preferred embodiments for carrying out the present technology will be described. In addition, embodiment described below shows an example of typical embodiment of this technique, and, thereby, the scope of this technique is not interpreted narrowly. The description will be made in the following order.

1. Nucleic acid electrophoresis cartridge, nucleic acid electrophoresis method, and nucleic acid electrophoresis cartridge manufacturing method according to the first embodiment of the present technology (1) Nucleic acid electrophoresis cartridge (2) Nucleic acid electrophoresis method (3) Nucleic acid electrophoresis 1. Manufacturing method for cartridge Nucleic acid electrophoresis cartridge, nucleic acid electrophoresis method, and nucleic acid electrophoresis cartridge manufacturing method according to the second embodiment of the present technology (1) Nucleic acid electrophoresis cartridge (2) Nucleic acid electrophoresis method (3) Nucleic acid electrophoresis Cartridge manufacturing method

1. Nucleic Acid Electrophoresis Cartridge, Nucleic Acid Electrophoresis Method, and Nucleic Acid Electrophoresis Cartridge Manufacturing Method According to First Embodiment of the Present Technology (1) Nucleic Acid Electrophoresis Cartridge FIG. 1 relates to the first embodiment of the present technology. It is a schematic diagram explaining the structure of the cartridge for nucleic acid electrophoresis, and the procedure of the nucleic acid electrophoresis method using this cartridge.

  In FIG. 1A, a cartridge for nucleic acid electrophoresis denoted by reference numeral 1 has a channel 11 formed on a substrate, and a positive electrode 12 and a negative electrode 13 respectively disposed at both ends of the channel 11. A liquid can be introduced into the channel 11, and a polymer gel 14 is disposed between the positive electrode 12 and the negative electrode 13 of the channel 11. Further, a power source V is connected to the positive electrode 12 and the negative electrode 13 so that a voltage can be applied / released to the liquid introduced into the channel 11. The channel 11 accommodates an intercalator that can be inserted into a nucleic acid and a buffer solution.

  The material of the substrate can be glass or various plastics (polypropylene, polycarbonate, cycloolefin polymer, polydimethylsiloxane).

  As the positive electrode 12 and the negative electrode 13, those comprising sputtered or vapor-deposited gold (Au) or platinum are preferably employed.

  The polymer gel 14 is an example of a blocking portion in the present technology. As will be described later, the polymer gel 14 is not particularly limited as long as it can block nucleic acids in a sample during electrophoresis. In addition, as the damming portion, porous membranes such as dialysis membranes, reverse osmosis membranes, semipermeable membranes, ion exchange membranes, or porous membranes such as cellulose, polyacrylonitrile, ceramic, zeolite, polysulfone, polyimide, palladium, etc. May be used. Hereinafter, in the present embodiment (the same applies to the second embodiment of the present technology), the polymer gel 14 is mainly described as an example of the damming portion in the present technology.

  As the polymer gel 14, polyacrylamide is suitably employed, more preferably polyacrylamide containing an anionic functional group, more preferably polyacrylamide containing an anionic functional group having an acid dissociation constant (pKa) of 1 to 5. Acrylamide is used. In the present embodiment, acrylamide means acrylamide or (meth) acrylamide.

  The anionic functional group is not particularly limited, but examples thereof include carboxylic acids such as acetic acid, propionic acid and butyric acid; polybasic acids such as oxalic acid and phthalic acid; hydroxy acids such as citric acid, glycolic acid and lactic acid; acrylic acid And unsaturated acids or unsaturated polybasic acids such as methacrylic acid; amino acids such as glycine, partial esters of phosphoric acid, partial esters of sulfuric acid, phosphonic acids, and sulfonic acids.

Specifically, for example, as carboxylic acid, formic acid (pKa: 3.55), acetic acid (pKa: 4.56), propionic acid (pKa: 4.67), butanoic acid (pKa: 4.63), pentene Acids (pKa: 4.68), hexanoic acid (pKa: 4.63), heptanoic acid (pKa: 4.66), palmitic acid (pKa: 4.64), stearic acid (pKa: 4.69), etc. Aliphatic monocarboxylic acid; succinic acid (pKa ₁ : 4.00, pKa ₂ : 5.24), glutaric acid (pKa ₁ : 4.13, pKa ₂ : 5.03), adipic acid (pKa ₁ : 4. 26, pKa ₂ : 5.03), pimelic acid (pKa ₁ : 4.31, pKa ₂ : 5.08), suberic acid (pKa ₁ : 4.35, pKa ₂ : 5.10), azelaic acid (pKa _{1: 4.39, pKa 2: 5.12} ) Malic acid _{(pKa 1: 3.24, pKa 2} : 4.71), terephthalic acid _{(pKa 1: 3.54, pKa 2} : 4.46) aliphatic or aromatic dicarboxylic acids, such as; crotonic acid (pKa : 4.69), unsaturated carboxylic acids such as acrylic acid (pKa: 4.26), methacrylic acid (pKa: 4.66); anisic acid (pKa: 4.09), m-aminobenzoic acid (pKa _{1) : 3.12, pKa 2: 4.74)} , m-, p- chlorobenzoic acid _{(pKa 1: 3.82, pKa 2} : 3.99), hydroxybenzoic acid (pKa _1: 4.08, pKa ₂ : Substituted benzoic acids such as: 9.96); polycarboxylic acids such as citric acid (pKa ₁ : 2.87, pKa ₂ : 4, 35, pKa ₃ 5.69) and derivatives thereof.

  The acrylamide monomer containing an anionic functional group is particularly preferably acrylamide alkanesulfonic acid. Examples of the sulfonic acid include styrene sulfonic acid (pKa = −2.8), m-aniline sulfonic acid (pKa = 3.74), p-aniline sulfonic acid (pKa = 3.23), and 2- (meth). Sulfone having a polymerizable unsaturated group such as acrylamido-2-alkyl (1 to 4 carbon atoms) propanesulfonic acid, more specifically 2-acrylamido-2-methylpropanesulfonic acid (pKa = -1.7). Examples include acids.

  The concentration (% by weight) of the anionic functional group in the polyacrylamide gel is preferably 0 to 30%.

  The intercalator is not particularly limited as long as it is capable of binding to a nucleic acid and has an anionic functional group, but preferably has an acid dissociation constant (pKa) of more than 0 and 3 or less. Here, the intercalator refers to a substance that is inserted into a double strand of a nucleic acid.

Examples of the anionic functional group contained in the intercalator include a carboxyl group (—COOH group), a mercapto group (—SH group), a phosphate group (—PO ₃ H), a phosphate group (—PO ₄ H ₂ ), sulfo group _(-SO 3 H), include sulfate groups _(-SO 4 H) or the like.

  The anionic functional group contained in the intercalator is particularly preferably a sulfo group. Examples of the intercalator containing a sulfo group include 9,10-anthraquinone-2,6-disulfonic acid, sodium anthraquinone-1-sulfonate, disodium anthraquinone-2,7-disulfonate, anthraquinone-1,5- Examples include disodium disulfonic acid and sodium anthraquinone-2-sulfonate.

  In addition, the pH of the buffer solution is not particularly limited, but is preferably 2 to 3, for example. It is also preferable that the pH of the buffer is 3-4. Furthermore, it is also suitable that the pH of the buffer is 4-5. Examples of the buffer solution are not particularly limited. For example, glycine-hydrochloric acid buffer solution (pH: 2.0 to 3.0), citrate buffer solution (pH: 3.0 to 6.0) Acetate buffer (pH: 3.0 to 5.5), citrate-phosphate buffer (pH: 2.5 to 7.0), MES (pH: 5.0 to 7.0), Bis- Tris (pH: 5.0-7.0) etc. are mentioned.

(2) Nucleic Acid Electrophoresis Method The procedure of a nucleic acid electrophoresis method using the cartridge 1 for nucleic acid electrophoresis will be described with reference to FIGS. The nucleic acid electrophoresis method here is a nucleic acid concentration purification method,

  First, a sample containing nucleic acid is introduced into a region 113 between the polymer gel 14 of the channel 11 and the negative electrode 13 (see FIG. 1B). At this time, the channel 11 is filled with a buffer solution for electrophoresis. An intercalator is also accommodated in the region 113 between the polymer gel 14 and the negative electrode 13 of the channel 11.

  At this time, an intercalator is inserted into the nucleic acid introduced into the region 113. Regarding the step of inserting the intercalator into the nucleic acid, the intercalator is inserted into the nucleic acid in advance, and then the nucleic acid may be introduced into the region 113, or the nucleic acid is introduced into the intercalator previously accommodated in the region 113 and inserted. You may let them.

  Here, the sample containing the nucleic acid used in the nucleic acid electrophoresis method according to the present embodiment includes, for example, pharynx, nasal cavity, vaginal swab, blood, plasma, serum, sputum, spinal fluid, urine, sweat, stool, etc. A liquid derived from a living body is used. In addition, a cell culture solution, a bacterial culture solution, a liquid in food, a liquid in soil, or the like is also used for the sample. Therefore, the sample solution may also contain various proteins, sugars and the like (hereinafter referred to as contaminants, which are also the same in the second embodiment) that are not necessary for nucleic acid analysis. Therefore, in order to analyze nucleic acid with high efficiency, it is required to separate the nucleic acid from impurities.

  In this regard, as a technique related to the present technology, for example, there is a method of performing electrophoresis under acidic conditions (for example, pH: 2 to 4) of a buffer solution or the like to separate nucleic acids and contaminants. However, in such a method, there is a concern that the electrophoresis speed of the nucleic acid is slow and it is difficult to concentrate the nucleic acid. In addition, if the contaminants contain sialic acid or other proteins with other low isoelectric points, the isoelectric point of the contaminants and the isoelectric point of the nucleic acid will be close to each other. There is also a concern that it is difficult to separate the nucleic acid and the contaminants even if it is done.

  On the other hand, the intercalator used in the present embodiment has an anionic functional group. The pKa of the nucleic acid into which such an intercalator is inserted decreases, and the isoelectric point of the nucleic acid also decreases. Therefore, the difference in isoelectric point between the nucleic acid and the contaminant can be increased, and the electrophoresis speed of the nucleic acid becomes faster than the electrophoresis speed of the contaminant. Therefore, in the electrophoresis according to this embodiment to be described later, it is possible to stably separate nucleic acids and contaminants.

  Here, the influence of the isoelectric point on the electrophoresis speed will be described in detail with reference to FIG. FIG. 2 shows each base constituting a nucleic acid (adenine (represented by A in FIG. 2), guanine (represented by G in FIG. 2)), thymine (represented by T in FIG. 2), cytosine (represented by T). In FIG. 2, it is represented by C.) A graph of pH-charge curves for each) is shown.

  As shown in FIG. 2, the isoelectric point here refers to the pH at which the charge becomes zero. In any base, the negative charge increases as the pH increases with respect to the isoelectric point. Therefore, a substance having a lower isoelectric point can increase the electrophoresis speed of nucleic acid. Here, as described above, by inserting an intercalator containing an anionic functional group into the nucleic acid, the pH-charge curve of each base is shifted to the acidic side, and the isoelectric point is lowered. Regarding the isoelectric point of each base, for example, the isoelectric point of cytosine (C) is higher than that of other bases, about 2.5 to 3, and the acid dissociation constant of the intercalator is particularly 3 or less. When the nucleic acid is inserted into the intercalator, the electrophoresis speed of the nucleic acid can be further increased.

  In addition, by using a buffer having a pH of 2 to 3, it becomes easier to suppress electrophoresis of proteins contained in contaminants, and it is possible to perform electrophoresis of nucleic acids. Therefore, nucleic acid and impurities can be separated more stably and the nucleic acid can be purified efficiently.

  The nucleic acid electrophoresis method / nucleic acid electrophoresis method according to this embodiment will be described again with reference to FIG. As described with reference to FIG. 1B, after nucleic acid is introduced into the region 113, a voltage is applied between the positive electrode 12 and the negative electrode 13 to apply a voltage to the buffer solution to perform electrophoresis of the nucleic acid. . The negatively charged nucleic acid is electrophoresed in the channel 11 toward the positive electrode 12. At this time, the polymer gel 14 blocks the movement of the nucleic acid and blocks the nucleic acid, so that the nucleic acid is concentrated at the interface of the polymer gel 14 and its vicinity (see FIG. 1C). The electrophoresis time is appropriately set according to the size of the channel 11 and may be, for example, about 10 seconds to 10 minutes. The “interface” of the polymer gel refers to a contact surface between the polymer gel 14 and the buffer solution filled on the region 113 side.

  Finally, the concentrated nucleic acid is recovered by aspirating the buffer solution near the polymer gel 14 in the region 113 with the micropipette 2 or the like (see FIG. 1D).

  Thus, according to the nucleic acid electrophoresis method according to this embodiment, an intercalator having an anionic functional group is inserted into the nucleic acid. Preferably, the acid dissociation constant of the intercalator is more than 0 and 3 or less. Therefore, the negative charge of the nucleic acid into which the intercalator is inserted is strengthened, and the isoelectric point of the nucleic acid is lowered. In particular, when the intercalator contains a functional group having a very low acid dissociation constant such as a sulfo group, the isoelectric point of the nucleic acid into which the intercalator is inserted is greatly reduced. Therefore, according to the nucleic acid electrophoresis method according to the present embodiment, the electrophoresis speed of the nucleic acid can be increased, and the nucleic acid can be concentrated and purified in a short time. In the present technology, inserting an intercalator into a nucleic acid refers to inserting an intercalator between complementary base pairs of a double-stranded nucleic acid, in other words, intercalating the intercalator. In the present technology, the double-stranded nucleic acid includes a nucleic acid that is originally composed of a single strand but is partially double-stranded by self-hybridization.

  Furthermore, according to the nucleic acid electrophoresis method according to the present embodiment, as described above, the difference in isoelectric point between the nucleic acid contained in the sample liquid and the contaminant is increased by lowering the isoelectric point of the nucleic acid. be able to. For this reason, it is possible to suppress electrophoresis of nucleic acids while performing electrophoresis for nucleic acids, and to stably separate nucleic acids and contaminants. That is, according to the nucleic acid electrophoresis method according to this embodiment, a nucleic acid with higher purity can be purified.

  In addition, by lowering the pH of the buffer solution introduced into the region 113 (for example, 2 to 3 etc.), the nucleic acid has a negative charge while the negative charge of the contaminant is weakened. Electrophoresis can be performed, and nucleic acids and contaminants can be separated with higher accuracy.

  Furthermore, by allowing the polymer gel 14 to contain an anionic functional group, the nucleic acid moves to the inside of the polymer gel 14 due to the electric repulsion between the negatively charged nucleic acid and the anionic functional group. Can be prevented. When the nucleic acid moves to the inside of the polymer gel 14 or further passes through the polymer gel 14 and moves to the region 112 on the positive electrode 12 side, the amount of the recovered nucleic acid is reduced. The anionic functional group preferably has an acid dissociation constant (pKa) of 1 to 5. The functional group having a lower pKa and a larger negative charge has a higher effect of preventing the nucleic acid from moving into the polymer gel 14 by electric repulsion.

  Furthermore, applying a reverse voltage to the positive electrode 12 and the negative electrode 13 for a short time when sucking the buffer solution in the vicinity of the polymer gel 14 in the region 113 is also effective in increasing the amount of nucleic acid recovered. By applying a reverse voltage for a short time during the recovery of the nucleic acid, the nucleic acid existing at the interface of the gel or dialysis membrane or the nucleic acid existing inside the gel near the interface is moved to the vicinity of the polymer gel 14 in the region 113, and the micropipette. 2 can be sucked and collected. The time for applying the reverse voltage is appropriately set according to the size of the channel 11, but may be about 1 to 10 seconds, for example.

(3) Method for Producing Cartridge for Nucleic Acid Electrophoresis The cartridge for nucleic acid electrophoresis according to this embodiment is obtained by gelling a monomer solution introduced into a channel 11 formed on a substrate into a predetermined shape by photopolymerization. It can be manufactured by forming a polymer gel 14 and housing an intercalator in the channel 11.

  The channel 11 can be formed on the substrate by, for example, wet etching or dry etching of a glass substrate layer, or nanoimprinting, injection molding, or cutting of a plastic substrate layer. One or more channels 11 can be formed on the cartridge 1.

  In the monomer solution, an acrylamide monomer is suitably employed, more preferably an acrylamide monomer containing an anionic functional group, and more preferably an acrylamide monomer containing an anionic functional group having an acid dissociation constant (pKa) of 1 to 5. Is used. Specifically, the monomer solution is particularly preferably acrylamide alkanesulfonic acid.

  The positive electrode 12 and the negative electrode 13 are preferably produced by sputtering or vapor deposition of gold (Au) or platinum (Pt). In this regard, some related art cartridges use platinum wires as electrodes. In this case, it is necessary to arrange the platinum wire in the cartridge for nucleic acid electrophoresis every time the experiment is performed, and the cartridge cannot be made disposable in view of the fact that the platinum wire is expensive. On the other hand, as described above, in the cartridge 1 for nucleic acid electrophoresis according to the present embodiment, the positive electrode 12 and the negative electrode 13 are prepared by sputtering or vapor deposition of gold, and the electrodes are prepared in advance in the cartridge 1. 1 can be handled easily. Further, such a cartridge 1 for nucleic acid electrophoresis can be made disposable as compared with the cartridge for nucleic acid electrophoresis according to the related art. Therefore, in the cartridge 1 for nucleic acid electrophoresis, sample contamination can be prevented when the sample is concentrated.

2. Nucleic acid electrophoresis cartridge, nucleic acid electrophoresis method, and nucleic acid electrophoresis cartridge manufacturing method according to second embodiment of the present technology

(1) Cartridge for Nucleic Acid Electrophoresis FIG. 1 shows the configuration of a cartridge for nucleic acid electrophoresis according to the second embodiment and the nucleic acid electrophoresis using the cartridge, as described in the first embodiment of the present technology. It is a schematic diagram explaining the procedure of a method. Here, first, the configuration of the cartridge for nucleic acid electrophoresis according to the second embodiment of the present technology will be described with reference to FIG.

  In FIG. 1A, a cartridge for nucleic acid electrophoresis denoted by reference numeral 1 has a channel 11 formed on a substrate, and a positive electrode 12 and a negative electrode 13 respectively disposed at both ends of the channel 11. A liquid can be introduced into the channel 11, and a polymer gel 14 is disposed between the positive electrode 12 and the negative electrode 13 of the channel 11. Further, a power source V is connected to the positive electrode 12 and the negative electrode 13 so that a voltage can be applied / released to the liquid introduced into the channel 11. Further, the channel 11 accommodates a compound that undergoes a dehydration condensation reaction with a carboxyl group of impurities in the sample solution containing nucleic acid, and a buffer solution. As described above, in the cartridge for nucleic acid electrophoresis according to this embodiment, the channel 11, the positive electrode 12, the negative electrode 13, and the polymer gel 14 are substantially the same as in the first embodiment of the present technology described above. belongs to. Therefore, in the cartridge for nucleic acid electrophoresis according to this embodiment, the compound and buffer solution that perform the dehydration condensation reaction will be mainly described.

  The compound that undergoes the dehydration condensation reaction is not particularly limited as long as it can undergo a dehydration condensation reaction with the carboxyl group of the protein of the contaminant in the sample containing the nucleic acid. The compound preferably has a first cationic functional group that undergoes a dehydration condensation reaction with a carboxyl group, and a second cationic functional group that exists in an ionic state during electrophoresis of nucleic acids. That is, it is preferable that some functional groups of the compound are electrophoresed in an ionic state even after a dehydration condensation reaction between the compound and a protein in a contaminant.

  The compound preferably contains one or more amino groups and is amide-bonded to a carboxyl group in a protein of a contaminant.

  Specific examples of the compound include N-hydroxysuccinimide (NHS), ethanolamine, and ethylenediamine.

  The channel 11 preferably contains the condensing agent for the dehydration condensation reaction. The condensing agent is preferably a carbodiimide compound. Specific examples of the condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl)- Examples include 4-methylmorpholinium chloride (DMT-MM), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC).

  Further, the channel 11 of the cartridge for nucleic acid electrophoresis preferably further contains a buffer solution. The pH of the buffer solution is not particularly limited, but is preferably 3-4. It is also preferable that the pH of the buffer is 4 to 5. It is also preferable that the pH of the buffer is 5-6. It is also preferable that the pH of the buffer solution is 6-7. Furthermore, it is also preferable that the pH of the buffer is 7-8. It is also preferable that the buffer solution does not contain a carboxyl group or an amino group. Examples of the buffer solution include glycine-hydrochloric acid buffer solution (pH: 2.0 to 3.0), citrate buffer solution (pH: 3.0 to 6.0), and acetate buffer solution (pH: 3.0 to 3.0). 5.5), citrate-phosphate buffer (pH: 2.5-7.0), Tris (pH: 7.2-9.0), MES (pH 3.0-7.0), bis- Examples include tris (pH 5.0 to 7.0), PBS (pH 5.0 to 8.0), and the like.

(2) Nucleic Acid Electrophoresis Method Next, the procedure of the nucleic acid electrophoresis method using the cartridge 1 for nucleic acid electrophoresis according to this embodiment will be described with reference to FIGS.

  First, a sample containing nucleic acid is introduced into a region 113 between the polymer gel 14 of the channel 11 and the negative electrode 13 (see FIG. 1B). At this time, the channel 11 is filled with a buffer solution for electrophoresis. Further, the compound is also accommodated in a region 113 between the polymer gel 14 and the negative electrode 13 of the channel 11.

  At this time, the sample liquid containing the nucleic acid introduced into the region 113 contains impurities, and the carboxyl group of the protein in the impurities undergoes a dehydration condensation reaction with the above compound. The step of subjecting the sample liquid and the compound to a dehydration condensation reaction may be performed by introducing the sample liquid into the compound previously stored in the region 113, or by reacting the compound and the sample liquid in advance. From this, the sample liquid may be introduced into the region 113.

  In this manner, the negative charge of the protein is weakened and the isoelectric point is increased by the dehydration condensation reaction between the contaminant protein contained in the sample solution and the compound. Thereby, according to the nucleic acid electrophoresis method according to the present embodiment, the difference in isoelectric point between the nucleic acid contained in the sample liquid and the contaminants can be determined as in the nucleic acid electrophoresis method according to the first embodiment of the present technology. It becomes possible to enlarge.

  Subsequently, the method for concentrating and purifying nucleic acid as shown in FIGS. 1 (C) and 1 (D) will be described. The method for electrophoresis and concentration and purification of nucleic acid according to the first embodiment of the present technology described above. And substantially the same.

  Thus, according to the nucleic acid electrophoresis method according to the present embodiment, the protein contained in the impurities in the sample solution containing the nucleic acid undergoes a dehydration condensation reaction with the above compound. Therefore, the negative charge of the protein that has undergone dehydration condensation reaction with the above compound is weakened, and the isoelectric point of the protein is increased. In particular, when the compound contains a plurality of cationic functional groups, the isoelectric point of a protein that has undergone a dehydration condensation reaction with the compound is significantly increased. Therefore, even when nucleic acid electrophoresis is performed, impurities are less likely to migrate from the negative electrode side to the positive electrode side. Thus, according to the nucleic acid electrophoresis method according to the present embodiment, the difference in isoelectric point between the nucleic acid and the contaminant can be increased. Contaminants can be stably separated. That is, according to the nucleic acid electrophoresis method according to this embodiment, a nucleic acid with higher purity can be purified.

  In addition, by reducing the pH of the buffer solution introduced into the region 113, it becomes possible to perform electrophoresis in a state where the nucleic acid has a negative charge and the contaminant does not have a negative charge. Thus, nucleic acids and contaminants can be separated with higher accuracy. That is, according to the nucleic acid electrophoresis method according to this embodiment, the concentration rate of nucleic acid can be further improved. In addition, according to the nucleic acid electrophoresis method according to the present embodiment, the concentration time can be shortened. By using such a nucleic acid electrophoresis method, the nucleic acid amplification reaction can be performed with high accuracy.

  In addition, when a protein in a contaminant reacts with N-hydroxysuccinimide to form an amide bond, a compound having another amine functional group such as ethanolamine is replaced with N-hydroxysuccinimide and binds to the protein. can do. Since the protein and N-hydroxysuccinimide are relatively weakly bonded, the compound having the other amine functional group can be appropriately selected.

(3) Method for Producing Cartridge for Nucleic Acid Electrophoresis The nucleic acid concentration and purification cartridge according to this embodiment is obtained by gelling a monomer solution introduced into a channel 11 formed on a substrate into a predetermined shape by photopolymerization. It can be produced by forming a polymer gel 14 and housing the compound in the channel 11. In addition, the molding of the channel 11 and the like are performed in substantially the same manner as the method for manufacturing the cartridge for nucleic acid electrophoresis according to the first embodiment of the present technology described above.

  As mentioned above, although each embodiment has been described, the intercalator described in the first embodiment and the dehydrating condensing agent described in the second embodiment may be used together in the present technology. That is, the nucleic acid may be bound to an intercalator, and the contaminating protein in the sample solution may be bound to N-hydroxysuccinimide or the like for electrophoresis to concentrate the nucleic acid. The combined use of an intercalator and a compound that undergoes a dehydration condensation reaction can increase both the removal efficiency of contaminants and the nucleic acid concentration efficiency, so that nucleic acids can be concentrated and concentrated in a shorter time and with higher efficiency. it can.

In addition, this technique can also take the following structures.
(1) A nucleic acid electrophoresis method including a procedure for electrophoresis of a nucleic acid into which an intercalator having an anionic functional group is inserted.
(2) The nucleic acid electrophoresis method according to (1), wherein the acid dissociation constant of the intercalator is greater than 0 and 3 or less.
(3) The nucleic acid electrophoresis method according to (1) or (2), wherein the functional group is a sulfo group.
(4) including a step of applying a voltage to both ends of a channel filled with a buffer solution, causing electrophoresis of the nucleic acid introduced at a predetermined position in the channel, and damaging the nucleic acid moving to the positive electrode end (1 The nucleic acid electrophoresis method according to any one of (1) to (3).
(5) The nucleic acid electrophoresis method according to any one of (4), wherein the pH of the buffer solution is 5 or less.
(6) The nucleic acid electrophoresis method according to any one of (5), wherein the pH of the buffer solution is 4 or less.
(7) The nucleic acid electrophoresis method according to any one of (6), wherein the pH of the buffer solution is 3 or less.
(8) A method for concentrating and purifying nucleic acid, comprising a step of electrophoresis of a nucleic acid into which an intercalator having an anionic functional group is inserted.
(9) A channel into which a liquid can be introduced is formed, a blocking portion for blocking a substance is disposed at a predetermined position of the channel, electrodes are disposed at both ends of the channel, and an anionic functional group is inserted into a nucleic acid. A cartridge for nucleic acid electrophoresis in which a possible intercalator and a buffer solution are accommodated between a damming portion and a negative electrode end.
(10) forming a polymer gel by photopolymerization of a monomer solution introduced into a channel formed on a substrate to form a polymer gel; and a nucleic acid having an anionic functional group in the channel A method for producing a cartridge for nucleic acid electrophoresis, comprising a step of accommodating an intercalator that can be inserted into the cartridge.

1. Manufacture of cartridges for nucleic acid electrophoresis
A channel having a width of 5 mm, a length of 60 mm, and a depth of 2 mm was formed on a PMMA substrate. A positive electrode and a negative electrode formed with platinum wires (diameter 0.5 mm, length 10 mm, Nilaco Co., Ltd.) were disposed at both ends of the channel. This channel was filled with an acrylamide solution (solution 2) prepared according to “Table 1” and “Table 2”. Photopolymerization of acrylamide was performed using a confocal laser scanning microscope (Olympus, FV1000) to form a polymer gel having a width of 5 mm and a length of 3 mm in the channel. Thereafter, the unpolymerized acrylamide solution was replaced with an electrophoresis buffer (1 × TBE).

2. Preparation of sample solution The sample solution (Example 1) to be introduced into the prepared cartridge for nucleic acid electrophoresis was adjusted under the conditions shown in Table 3. Salmon sperm (final concentration: 0.1 mg / mL) was used as the nucleic acid. As the buffer, glycine hydrochloride buffer (pH: 2.2) (final concentration: 50 mM) was used. Anthraquinone-2,6-disulfonate disodium (final concentration: 1 mM) was used as an intercalator. SYBR (registered trademark) GREENI was used as a fluorescent reagent. In addition, as Comparative Example 1, a sample solution prepared under the same conditions as Example 1 was prepared except that distilled water was used instead of using anthraquinone-2,6-disulfonic acid disodium.

  Further, as Example 2, as compared with Example 1, instead of using glycine hydrochloride buffer (pH: 2.2) (final concentration: 50 mM), acetate buffer (pH: 4.0) (final concentration: 50 mM) A sample solution prepared under the same conditions except that was used was also prepared. Further, as Comparative Example 2, as compared with Example 2, a sample solution prepared under the same conditions except that distilled water was used instead of using anthraquinone-2,6-disulfonic acid disodium was also prepared.

3. Nucleic acid electrophoresis (1)
Nucleic acid was concentrated using the prepared cartridge for nucleic acid electrophoresis. 500 μl of the prepared sample solutions (four kinds of sample solutions prepared under the conditions of Examples 1 and 2 and Comparative Examples 1 and 2) were introduced between the polymer gel of the channel and the negative electrode side. A voltage was applied to the electrode, electrophoresis was performed at 75 V and 1 mA, and the moving sample was observed with a confocal laser scanning microscope.

  FIG. 3 is a sample of Example 1 (shown as pH 2.2 Anthraquinone-2,6-disulfonate in the figure) and Comparative Example 1 (shown as pH 2.2 negative control in the figure). It is a graph which shows a time-dependent change of the fluorescence intensity of the gel interface after the electrophoresis start in the case of using a liquid. In FIG. 3, the horizontal axis represents time (minutes), and the vertical axis represents fluorescence intensity. 4 shows Example 2 (shown as pH 4.0 Anthraquinone-2,6-disulfonate in the figure) and Comparative Example 2 (shown as pH 4.0 negative control in the figure). It is a graph which shows a time-dependent change of the fluorescence intensity of the gel interface after the start of electrophoresis in the case of using this sample liquid. In FIG. 4, as in FIG. 3, the horizontal axis represents time (minutes) and the vertical axis represents fluorescence intensity.

  As shown in FIG. 3, in the case of using a buffer solution of pH 2.2, in Example 1 using an intercalator, compared to Comparative Example 1 not using an intercalator, 1 minute after the start of electrophoresis of nucleic acids, A concentration factor of about 2 times could be realized. In addition, as shown in FIG. 4, even when a pH 4.0 buffer solution was used, in Example 2 using an intercalator, compared to Comparative Example 1 using no intercalator, 1 minute of starting electrophoresis of nucleic acid. Later, a concentration factor of about 2 was achieved. Thus, it was demonstrated that a high concentration ratio of nucleic acid in an acidic buffer solution can be realized in a short time by inserting an intercalator having a negative charge into the nucleic acid. It was also demonstrated that by increasing the electrophoresis speed of nucleic acids, it is possible to increase the difference in electrophoresis speed between nucleic acids and proteins in contaminants, and to stably separate nucleic acids and contaminants. .

  After the completion of electrophoresis, a reverse voltage (75 V, 1 mA) was applied to the electrode for 10 seconds to release the concentrated sample from the gel interface. A micropipette cartridge with negative pressure inside was brought into contact with the gel interface, and 10 μl of the concentrated sample was collected.

4). Nucleic acid electrophoresis (2)
Here, as Examples 3 and 4, changes in the isoelectric point of the protein due to isoelectric focusing after the protein was dehydrated and condensed were analyzed. As the protein, Bovine Serum Albumin (BSA) (pI = 4.9) 1 mg / mL (Example 3) or α1-acid glycoprotein (AGP) (pI = 2.7) 1 mg / mL (Example 4) was used. NHS 5mM, EDC 20mM, ethanolamine 50mM, or ethylenediamine 50mM was used as a substance necessary for the dehydration condensation reaction. As the buffer, TBE (pH 8.5), MES (pH 4.0), MES (pH 5.0), Bis-Tris (pH 6.5), or Bis-Tris (pH 5.5) was used.

  Incubation was performed at room temperature for 1 hour. Then, 10 uL of the sample subjected to the dehydration condensation reaction (Examples 3 and 4), 1 mg of protein BSA not subjected to the dehydration condensation reaction as negative control, 1 mg of AGP (Comparative Examples 3 and 4), and IEF Marker pI 3-10 (Invitrogen) ) Was subjected to isoelectric focusing.

  FIG. 5 shows the results of isoelectric focusing using BSA (Example 3). FIG. 6 shows the results of isoelectric focusing using AGP (Example 4).

  As shown in FIGS. 5 and 6, a protein band is observed at a predetermined position under a negative control in which dehydration condensation reaction is not performed. On the other hand, in the sample subjected to the dehydration condensation reaction, the concentration of the band at the predetermined position is decreased compared to the protein not subjected to the dehydration condensation reaction, or the band position is moved to the upper part of the gel. There were some gels of isoelectric focusing that could not be observed without electrophoresis. From these results, it was demonstrated that the isoelectric point of the protein was increased by the dehydration condensation reaction. That is, it was demonstrated that the difference in electrophoresis speed between the nucleic acid and the protein in the contaminant can be increased, and the nucleic acid and the contaminant can be stably separated.

  When electrophoresis is performed with an electrophoresis buffer (pH: 6.0 to 9.0) used for normal nucleic acid electrophoresis, the sample subjected to the dehydration condensation reaction has a positive charge and is migrated to the cathode. On the other hand, a nucleic acid that has not undergone dehydration condensation reaction has a negative charge, so that it can be purified by being migrated to the anode. In particular, by setting the pH of the electrophoresis buffer to 6.0 or less, preferably 5.0 or less, more preferably further to the acidic side, proteins that have not undergone dehydration condensation can be separated from nucleic acids, The purity of the nucleic acid can be increased.

  The nucleic acid electrophoresis method according to the present technology is simple in operation, and can concentrate and purify nucleic acids with high efficiency in a short time. Therefore, it is applied to nucleic acid concentration treatment for nucleic acid amplification reaction such as PCR (Polymerase Chain Reaction) method and LAMP (Loop-Mediated Isothermal Amplification) method. Can be used to detect.

1: cartridge, 2: micropipette, 11: channel, 12: positive electrode, 13: negative electrode, 14: polymer gel

Claims (10)

  A nucleic acid electrophoresis method comprising a procedure for electrophoresis of a nucleic acid having an intercalator having an anionic functional group inserted therein.   The nucleic acid electrophoresis method according to claim 1, wherein the acid dissociation constant of the intercalator is greater than 0 and 3 or less.   The nucleic acid electrophoresis method according to claim 2, wherein the functional group is a sulfo group. A voltage is applied to both ends of the channel filled with the buffer solution, and the nucleic acid introduced into a predetermined position in the channel is electrophoresed,
The nucleic acid electrophoresis method according to claim 1, comprising a step of damaging the nucleic acid moving to the positive electrode end.   The nucleic acid electrophoresis method according to claim 4, wherein the buffer solution has a pH of 5 or less.   The nucleic acid electrophoresis method according to claim 5, wherein the buffer solution has a pH of 4 or less.   The nucleic acid electrophoresis method according to claim 6, wherein the buffer solution has a pH of 3 or less.   A method for concentrating and purifying nucleic acid, comprising a step of electrophoresis of a nucleic acid into which an intercalator having an anionic functional group is inserted.   A channel into which a liquid can be introduced is formed, a blocking portion for blocking the substance is disposed at a predetermined position of the channel, electrodes are disposed at both ends of the channel, and an interface having an anionic functional group that can be inserted into a nucleic acid is provided. A cartridge for nucleic acid electrophoresis in which a calator and a buffer solution are accommodated between a damming portion and a negative electrode end. Forming the polymer gel by gelling the monomer solution introduced into the channel formed on the substrate into a predetermined shape by photopolymerization;
A method for producing a cartridge for nucleic acid electrophoresis, comprising: an intercalator having an anionic functional group in a channel and capable of being inserted into a nucleic acid.