JP5760206B2 - Method for detecting cleavage of nucleic acid contained in a single cell - Google Patents

Description

Cross-reference of related applications

This application claims the priority of Japanese Patent Application No. 2012-166668 filed on July 27, 2012, the entire description of which is incorporated herein by reference.

The present invention relates to a method for detecting cleavage of a nucleic acid contained in one cell, a method for producing a cell in which nucleic acid is not cleaved, and a kit for carrying out these.

There are various types of DNA damage, such as double-strand breaks, one-side cleavage, oxidative damage, and base modification by chemicals. Among them, double-strand breaks are the most difficult to repair endogenously. If it is damaged, even a single cut will be a serious problem, so it is the most lethal to cells.

Although somatic cells are equipped with multiple DNA repair mechanisms, sperm lose their DNA repair ability after the second meiotic late stage. If sperm with unrepaired or incompletely repaired DNA fertilizes with an egg, it leads to pregnancy loss such as chromosomal abnormalities and increased miscarriage rates. Therefore, the allowable limit of DNA double-strand break is extremely low, and from the viewpoint of sperm quality control in assisted reproduction (ART), establishment of a method capable of detecting the initial stage of DNA break is desired.

Known methods for detecting DNA cleavage include the sperm chromatin diffusion test, the TUNEL method, and the COMET method.

In the sperm chromatin diffusion test, sperm embedded in agarose is lysed, and then a DNA fragment diffused around is observed (see Non-Patent Document 1 below, the description of which is incorporated herein by reference) ). In the TUNEL method, a DNA fragment is labeled with a fluorescent dye and observed with a microscope (see Non-Patent Documents 2 and 3 below, the description of which is incorporated herein by reference).

The most widely used method for detecting DNA breakage in a single cell is the COMET method, and many papers using the method have been published (see Non-Patent Documents 4 to 6 below, The description of this document is incorporated herein by reference). In the COMET method, sperm embedded in agarose is lysed and then DNA fragments are separated by electrophoresis. In the COMET method, DNA double-strand breaks and one-side cleavage can be observed by alkali treatment.

Fernandez JL, Muriel L, RiveroMT, Goyanes V, VazquezR, et al. (2003) The sperm chromatin dispersion test: asimple method for the determination of sperm DNA fragmentation. JAndrol 24: 59-66. Dominguez-Fandos D, Camejo MI, Ballesca JL, Oliva R (2007) Human sperm DNA fragmentation: correlation of TUNEL results as correlated by flow cytometry and optical microscopy. CytometryPart A 71A: 1011-1018. Sharma RK, Sabanegh E, Mahfouz R, Gupta S, Thiyagarajan A, et al. (2010) TUNEL as a test for sperm DNA damage in the evaluation of male infertility. Urology 76: 1380-1386. Hughes CM, McKelvey-Martin VJ, Lewis SE (1999) Human sperm DNA integrity condensed by the Comet and ELISAassays. Mutagenesis 194: 71-75. Morris ID, Ilott S, Dixon L, Brison DR (2002) The spectrum of DNA damage in human sperm representing by singlecell gel electrophoresis (Comet assay) and its relationship to fertilizationand embryo development.Hum Reprod 17: 990-998. Simon L, Lutton D, McManus J, Lewis SE (2011) Sperm DNA damage measured by the alkaline Comet assay as anindependent predictor of male infertility and in vitro fertilization success. Fertil Steril 95: 652-657.

However, the sperm chromatin diffusion test, the TUNEL method and the COMET method are all insufficient as methods capable of detecting the initial stage of DNA cleavage.

That is, the sperm chromatin diffusion test detects a state in which fragmentation has progressed, and cannot detect the initial stage of DNA cleavage. In the TUNEL method, in order to sense fluorescence, a certain amount of DNA fragment needs to be labeled with a chromophore. The TUNEL method is also unsuitable for detecting the initial stage of DNA cleavage.

Furthermore, since the COMET method detects particulate fragments generated by highly DNA fragmentation, it cannot detect several cleavages of DNA contained in a single cell. For this reason, the COMET method does not have sufficient sensitivity as a method capable of detecting the initial stage of DNA cleavage.

In view of the problems of the prior art, an object of the present invention is to provide a new method capable of detecting the initial stage of DNA cleavage.

In eukaryotic cells, intracellular DNA is bound to nuclear proteins and is further fixed to the nuclear membrane with a structural protein called a nuclear matrix. In prokaryotes, DNA is bound to nucleoid proteins.

As a result of diligent research, the inventor of the present invention (a) embeds and solidifies a cell in a melt gel to which a proteolytic enzyme that has been previously inactivated has been added, and then activates the proteolytic enzyme in the gel to Cell nucleoside DNA can be separated from nucleoprotein and nuclear matrix, and (b) the separated intracellular DNA is electrophoresed by pulse field electrophoresis to one cell. It has been found that a long-chain DNA fragment found at an early stage of the DNA cleavage process contained in the cell nucleus can be detected, and has solved the above problems.

In particular, the present inventor studied to detect various long-chain DNA fragments with higher sensitivity. First, the present inventor has paid attention to the fact that the length of a DNA fragment obtained by one-dimensional pulse field electrophoresis is shorter than the length of intracellular DNA. Therefore, it was speculated that there might be long DNA fragments that could not be observed by one-dimensional pulse field electrophoresis. Therefore, an attempt was made to carry out the pulse field electrophoresis in two dimensions, not in one dimension. However, when the experimental method of one-dimensional pulse field electrophoresis was simply applied to the two-dimensional pulse field electrophoresis, the observation result of electrophoresis did not change.

Therefore, the present inventor determined the agarose concentration, the composition of the cell lysate, the angle of the two electrodes, the electrophoresis voltage, the electrode switching timing, the composition of the electrophoresis solution, the rotation angle of the slide glass smeared with the cells, etc. As a result of diligent investigations on various conditions, we succeeded in detecting long-chain DNA fragments that could not be detected by one-dimensional pulse field electrophoresis by two-dimensional pulse field electrophoresis. This is a surprising fact found for the first time by the inventor based on the inventor's guess. The present invention has been completed based on the above findings.

That is, according to the present invention, there is provided a method for detecting cleavage of a nucleic acid contained in one cell, comprising the following steps (1) to (5).
(1) Step of placing one or a plurality of cells on a support having an amino group on the surface (2) A mixture of a purified proteolytic enzyme having an optimum pH weakly alkaline and a weakly acidic solution containing a gelling agent (3) solidifying the weak acid solution (4) solidifying the weak acid solution into a weak alkaline solution containing a condensed phosphate compound, a surfactant and a reducing agent. Step of soaking (5) Step of separating nucleic acid derived from cells on a support soaked in the weak alkaline solution by pulse field electrophoresis using an electrophoretic solution containing a condensed phosphate compound

According to another aspect of the present invention, there is provided a method for producing a cell whose nucleic acid has not been cleaved, comprising the following steps (1) to (6).
(1) A step of placing one or a plurality of cells in a test sample on a support having an amino group on the surface (2) A weakly acidic solution comprising a purified proteolytic enzyme having a weakly alkaline optimum pH and a gelling agent The step of covering the cells with a mixture with the solution (3) The step of solidifying the weakly acidic solution (4) The support on which the weakly acidic solution is solidified comprises a condensed phosphate compound, a surfactant and a reducing agent. Step of immersing in weak alkaline solution (5) Step of separating nucleic acid derived from cells on a support immersed in the weak alkaline solution by pulse field electrophoresis using an electrophoresis solution containing a condensed phosphate compound (6) A step of determining a cell in the test sample as a cell whose nucleic acid has not been cleaved when the separation mode of the electrophoresed nucleic acid is similar to the separation mode of a nucleic acid derived from a normal cell.

In the present invention, preferably, the purified proteolytic enzyme having an optimum pH that is weakly alkaline has an activity of decomposing a peptide bond between arginine and any amino acid and / or a peptide bond between lysine and any amino acid.

In the present invention, preferably, the purified proteolytic enzyme whose optimum pH is weakly alkaline is a purified trypsin-like enzyme.

In the present invention, preferably, the condensed phosphoric acid compound is at least one condensed phosphoric acid compound selected from the group consisting of polyphosphoric acid, hexametaphosphoric acid and salts thereof.

In the present invention, preferably, the pulse field electrophoresis has two or more sets of electrodes, and the two or more sets of electrodes intersect at an angle of 45 to 120 degrees. Is achieved.

In the present invention, preferably, in the pulse field electrophoresis, after the first pulse field electrophoresis is performed, the support is rotated by 60 to 160 degrees with the cell arrangement portion as the rotation center, and the second pulse field electrophoresis is performed. This is accomplished by performing electrophoresis.

In the present invention, preferably, the cell is a sperm.

According to another aspect of the present invention, there is provided a kit for detecting nucleic acid cleavage or for producing a cell in which a nucleic acid is not cleaved, comprising the following components (1) to (5): .
(1) Support having an amino group on the surface (2) Purified proteolytic enzyme having an optimum pH weakly alkaline (3) Weakly acidic solution containing a gelling agent (4) Condensed phosphate compound, surfactant and reduction Weak alkaline solution containing an agent (5) Electrophoretic solution containing a condensed phosphate compound

In the present invention, the following component (6) is preferably further included.
(6) Electrophoresis device having two or more electrodes and the two or more electrodes intersecting at an angle of 45 to 120 degrees

According to the present invention, the double-stranded DNA constituting the chromosome can be observed as a DNA fiber, and when the double-stranded DNA in the cell is cut, it is cut according to the degree of the cut. It can be observed as a filamentous fragment or a particulate fragment.

That is, according to the present invention, the number of filamentous fragments excised by cutting one to several nucleic acids contained in one cell and the shortening of filamentous fragments by increasing the number of cleavage points, and further fragmentation are highly advanced. Particulate fragments generated by progressing can be detected.

Therefore, the present invention has an extremely high sensitivity that can be detected even at one to several cleavages of nucleic acid contained in one cell. Therefore, in the present invention, DNA cleavage that could not be detected by the conventional COMET method was detected. An initial stage can be detected.

FIG. 1A is a photograph showing a sperm head when purified motor sperm is suspended in a cell lysate. No voltage is applied. FIG. 1B is a photograph showing a sperm head when purified trypsin is added to the suspension when purified motor sperm is suspended in a cell lysate. No voltage is applied. FIG. 2A is a photograph showing DNA when trypsin is added to purified motor sperm embedded in agarose. No voltage is applied to the DNA of FIG. 2A. FIG. 2B is a photograph showing DNA when trypsin is added to ejaculated semen embedded in agarose. No voltage is applied to the DNA of FIG. 2B. FIG. 2C is a photograph showing DNA when pulsed field electrophoresis was performed on purified motile spermatozoa lysed in the presence of trypsin. FIG. 2D is a photograph showing DNA when ejaculated semen cell-lysed in the presence of trypsin is subjected to pulse field electrophoresis. FIG. 3A is a photograph showing an enlarged view of a DNA fiber stretched by pulse field electrophoresis. In this DNA, the DNA fiber is not cut. FIG. 3B is a photograph showing an enlarged view of a tip portion of a DNA fiber stretched by pulse field electrophoresis. In this DNA, the DNA fiber is not cut. FIG. 4A is a photograph showing an enlarged view of a DNA fiber stretched by pulse field electrophoresis. In these DNAs, several filamentous fragments and particulate fragments are observed at the tip of the DNA fiber. FIG. 4B is a photograph showing an enlarged view of a DNA fiber stretched by pulse field electrophoresis. In these DNAs, several filamentous fragments and particulate fragments are observed at the tip of the DNA fiber. FIG. 4C is a photograph showing an enlarged view of a DNA fiber stretched by pulse field electrophoresis. In these DNAs, several filamentous fragments and particulate fragments are observed at the tip of the DNA fiber. FIG. 5A is a photograph showing DNA when pulsed field electrophoresis is performed after cell lysis without adding trypsin in the case of purified motile sperm. FIG. 5B is a photograph showing DNA when pulse field electrophoresis is performed after cell lysis without adding trypsin in the case of ejaculate. FIG. 6A is a photograph showing DNA obtained by subjecting purified motor spermatozoa treated with trypsin to pulse field electrophoresis after alkali treatment. It was shown that DNA cleavage was artificially induced by alkali treatment. FIG. 6B is a photograph showing DNA obtained by subjecting purified motor spermatozoa treated with trypsin to pulse field electrophoresis after alkali treatment. It was shown that DNA cleavage was artificially induced by alkali treatment. FIG. 6C is a photograph showing DNA obtained by subjecting purified motor sperm treated with trypsin to alkali treatment after pulse field electrophoresis. It was shown that DNA cleavage was artificially induced by alkali treatment. FIG. 6D is a photograph showing DNA obtained by subjecting purified motor spermatozoa treated with trypsin to alkali treatment after pulse field electrophoresis. It was shown that DNA cleavage was artificially induced by alkali treatment. FIG. 7 is a photograph showing an enlarged view of a DNA fiber stretched by two-dimensional pulse field electrophoresis. Arrows (→) indicate long DNA fragments that cannot be observed by one-dimensional pulse field electrophoresis. In the COMET method, DNA strands are cleaved by a strong alkali treatment to detect nicks. FIG. 8 is a schematic diagram of DNA fibers stretched by one-dimensional pulse field electrophoresis (upper side) and two-dimensional pulse field electrophoresis (lower side).

1. The present invention relates to a method for detecting cleavage of a nucleic acid contained in one cell. Furthermore, one specific aspect of the present invention relates to a method for detecting cleavage of a nucleic acid contained in one cell, comprising the following steps (1) to (5).
(1) Step of placing one or a plurality of cells on a support having an amino group on the surface (2) A mixture of a purified proteolytic enzyme having an optimum pH weakly alkaline and a weakly acidic solution containing a gelling agent (3) solidifying the weak acid solution (4) solidifying the weak acid solution into a weak alkaline solution containing a condensed phosphate compound, a surfactant and a reducing agent. Step of soaking (5) Step of separating nucleic acid derived from cells on a support soaked in the weak alkaline solution by pulse field electrophoresis using an electrophoretic solution containing a condensed phosphate compound

In the present invention, the “cell” may be prepared or isolated from a sample derived from any species. As a sample containing such cells, a prokaryotic or eukaryotic individual itself or a part thereof can be used. For example, in vertebrates (including humans), excreta such as feces, urine, or sweat, body fluids such as blood, semen, saliva, stomach fluid, or bile, biopsy samples, and the like can be mentioned.

In addition, when using the cell which is not isolate | separated from a structure | tissue, a cell can also be isolate | separated from a structure | tissue, for example by performing a collagenase process.

Non-limiting examples of “cells” include fibroblasts, nerve cells, somatic cells such as blood cells (such as T cells), and germ cells such as sperm, preferably germ cells, more preferably Is a sperm.

In the present invention, “nucleic acid” refers to DNA and RNA contained in a cell (for example, cell nucleus), preferably DNA, and more preferably intracellular DNA. In addition, intracellular DNA is synonymous with chromosomal DNA or genomic DNA.

In the present invention, “nucleic acid cleavage” refers to cleavage of double-stranded nucleic acid and single-stranded nucleic acid.

Hereinafter, each step of the “method for detecting cleavage of nucleic acid contained in one cell” of the present invention will be described in detail.

(1) In the step (1) of placing one or more cells on a support having an amino group on the surface, one or more cells are placed on a support having an amino group on the surface.

The support is a support disposed in the electrophoresis apparatus when performing the pulse field electrophoresis in the step (5). The support is not particularly limited as long as it has an amino group on its surface, and examples thereof include a slide glass having an amino group on its surface. The method for imparting amino groups to the surface of the support is not particularly limited, and the method of treating the surface of the support with an amino group-containing compound or the compound having an amino group as a constituent component may be used to orient the amino groups on the surface. In addition, a technique for expressing a specific functional group on the surface of the support known by those skilled in the art, such as a technique for producing a support, can be employed.

The method for arranging the cells on the support is not particularly limited. For example, a method of dropping a solution containing cells onto the support and then fixing the cells on the support by centrifugal action can be employed.

(2) In the process step (2) in which the cells are covered with a mixture of a purified proteolytic enzyme whose optimum pH is weakly alkaline and a weakly acidic solution containing a gelling agent, a purified protein whose optimum pH is weakly alkaline The cells are covered with a mixture of a degrading enzyme and a weakly acidic solution containing a gelling agent.

In the present invention, the proteolytic enzyme is capable of separating nucleic acids contained in cells from nuclear proteins and / or nuclear matrices by degrading proteins.

The proteolytic enzyme is not particularly limited as long as it is purified and the optimum pH is weakly alkaline. For example, a peptide bond between arginine and any amino acid and / or lysine and any amino acid. And an enzyme having an activity of breaking the peptide bond. This is because the main nuclear proteins histone and protamine are rich in arginine and lysine. Since the positive charge of these basic residues in the nucleoprotein and the negative charge of the phosphate group of DNA form an ionic bond, an enzyme that functions to dissociate this ionic bond is preferred. Preferred examples of the proteolytic enzyme are pancreatic trypsin derived from various animals, trypsin-like proteolytic enzyme having various substrate specificities similar to those of various animals and microorganisms, and a widely used proteolytic enzyme (Proteinase K). More preferred is trypsin. Trypsin can maintain its purity and activity under acidic conditions, for example, under pH 2.0 or lower.

In the present invention, a purified protease is used. The degree of purification of the proteolytic enzyme is not particularly limited as long as it shows an activity to dissociate intracellular nuclear protein and DNA. For example, the purity of the proteolytic enzyme is 50% or more, preferably 60% or more, More preferably, it is 70% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more. The method for purifying the proteolytic enzyme is not particularly limited, and a general protein purification method known to those skilled in the art can be adopted. For example, a column on which a substance having high affinity for the proteolytic enzyme is immobilized is used. The affinity chromatography method used can be used.

In the present invention, the proteolytic enzyme has a weakly alkaline optimum pH. The optimum pH is as commonly understood in the art, and can be said to be, for example, a pH at which a proteolytic enzyme exhibits a relatively high activity. The optimal pH of the proteolytic enzyme is weakly alkaline, for example, pH 7.0 to 12.0, preferably pH 7.5 to 10.0, and more preferably pH 7.8 to 9.0.

Prokaryotes do not have nucleoproteins and / or nuclei matrix, but have nucleoid proteins. Therefore, in the present invention, the proteolytic enzyme applied to prokaryotes can separate nucleic acids contained in cells from nucleoid proteins, and for example, trypsin or proteinase K can be used.

The proteolytic enzyme used in the present invention may be a protein isolated from any biological species. Alternatively, the protein may be a chemically synthesized or biochemically synthesized protein in a cell-free translation system, or a recombinant produced from a transformant introduced with a nucleic acid having a base sequence encoding a proteolytic enzyme. It may be a protein.

In the present invention, as the proteolytic enzyme, not only a single proteolytic enzyme but also a mixture of a plurality of proteolytic enzymes can be used.

As the gelling agent used in the present invention, a known gelling agent can be used. For example, agarose, gelatin, starch, carrageenan, pectin, agaropectin, polyacrylamide, polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, Alternatively, trehalose or the like can be used, but agarose that forms a stable three-dimensional network structure is preferable. This is because agarose can form a stable three-dimensional network structure without using a factor that may adversely affect DNA, such as an oxidizing agent or ultraviolet rays.

The agarose concentration is set to a low concentration in consideration of the length of DNA in the cell nucleus. For example, the concentration (final concentration) of agarose is 0.1 to 1.0 w / v%, preferably 0.1 to 0.6 w / v%, more preferably 0.2 to 0.5 w / v. v%, more preferably 0.2 to 0.3 w / v%.

In the present invention, a solubilizer for solubilizing the cell membrane may be added to the weakly acidic solution.

Examples of the solubilizer used in this step include a surfactant. As the surfactant, those having a milder action on proteolytic enzymes are preferable. Examples of the surfactant include digitonin, saponin, Triton X100, Triton X114, Tween 20, and Tween 80. Any solubilizer is commercially available.

The pH range of the weakly acidic solution is, for example, pH 3.0 to 7.0, preferably pH 4.0 to 6.0. The method for adjusting the weakly acidic solution to be weakly acidic is not particularly limited, and can be achieved, for example, by containing a certain concentration of acid or buffer adjusted to be weakly acidic.

Other conditions such as the concentration of the solution in step (2), the treatment temperature, and the treatment time vary depending on the type of cells and the types of inclusions such as proteolytic enzymes, gelling agents, and solubilizing agents. Conditions can be set as appropriate. There is no particular limitation on the method of covering the cells placed on the support with a mixture of a purified proteolytic enzyme whose pH is weakly alkaline and a weakly acidic solution containing a gelling agent. For example, the cells on the support are covered with the mixture. It can be carried out by dripping onto the smeared part.

Further, in step (2), the pH is preferably adjusted so as to be neutral to slightly acidic so that the pH does not become 10 or more. This is because, as will be described later, fragmentation of nucleic acid is induced by alkali treatment. The step (2) is preferably carried out at a relatively high temperature condition that can maintain the gelling agent in a molten state, for example, 30 ° C. or higher. However, if the temperature is too high, the enzyme is deactivated and the nucleic acid is denatured. There is a probability.

(3) In the step (3) in which the weakly acidic solution in the step (2) is solidified, the weakly acidic solution in the step (2) is solidified.

For example, it can be solidified by adding a proteolytic enzyme and a gelling agent to a heated liquid and returning the obtained solution to room temperature (for example, 10 to 30 ° C.). When chemical polymerization is required, a polymerization initiator may be added according to the properties of the gelling agent.

By the steps (2) and (3), nucleic acids such as intracellular DNA are embedded in the gel. Step (3) is performed under conditions where the proteolytic enzyme is inactive, for example, under weak acid conditions.

(4) In the step (4) in which the support obtained by solidifying the weakly acidic solution in the step (3) is immersed in a weak alkaline solution containing a condensed phosphate compound, a surfactant, and a reducing agent, the step (3) ), The support obtained by solidifying the weakly acidic solution is immersed in a weakly alkaline solution that is a cell lysate.

The weak alkaline solution is not particularly limited as long as it contains a condensed phosphate compound, a surfactant, and a reducing agent, and can further contain, for example, a buffer and other components. A condensed phosphoric acid compound is a compound in which two or more phosphoric acids are reacted, water molecules are eliminated, and one new molecule obtained (H _{n + 2} P _n O _{3n + 1} (n is an integer of 1 or more)) Or a salt thereof.

The reason for adding the condensed phosphate compound to the cell lysate is as follows. That is, the DNA has a structure in which two nucleobases are bound to a main chain in which deoxyribose (pentose sugar) and phosphoric acid phosphodiester bonds are repeated, and the two are bound in a spiral. The phosphate group in the phosphodiester bond has a strong negative charge. On the other hand, since arginine and lysine constituting the nuclear protein have a positive charge, the nuclear protein and phosphate are electrostatically coupled. A condensed phosphate compound can efficiently dissociate DNA by competitively binding to a nuclear protein. Thus, since the condensed phosphate compound can specifically competitively dissociate the binding between the nucleoprotein and the DNA, the concentration thereof can be a low concentration, for example, a concentration of 10 mM or less.

Conventionally, high-concentration salts (such as NaCl of 2 M or more) have been used for dissociation and solubilization of DNA and nucleoprotein. The inventor noticed that a small amount of condensed phosphate compounds such as hexametaphosphoric acid and polyphosphoric acid dissociates the DNA-nucleoprotein complex, and for the first time, this was adopted in the DNA fragmentation test. Suitable examples of the condensed phosphoric acid compound are hexametaphosphoric acid and its salt and polyphosphoric acid and its salt.

By the way, the steps (2) to (4) of the present invention are supported by the cell lysate containing the proteolytic enzyme after solidifying the weakly acidic solution containing the gelling agent without containing the proteolytic enzyme in the weakly acidic solution. It can be considered to be a step of immersing the body. However, it is difficult to treat cells embedded in the gel with a proteolytic enzyme added thereafter. After the gel is solidified, low molecular weight reagents (DTT, salts, etc.) diffuse easily in the gel network, but proteolytic enzymes with molecular weights of tens of thousands or more are not easy to diffuse in the gel. Because most do not reach around the cells.

Steps (2) to (4) overcome such difficulties. In the step (3), the weakly acidic solution containing the proteolytic enzyme and the gelling agent in the step (2) is solidified. Through these steps, the proteolytic enzyme is uniformly diffused in the solidified gel.

Importantly, steps (2) and (3) are performed under conditions where the proteolytic enzyme is inactive, and step (4) is performed under conditions where the proteolytic enzyme is active. . This is because if a cell is thawed by a proteolytic enzyme before the gel is solidified, DNA diffuses into the solution and it is difficult to determine a DNA fragment derived from a single cell. Accordingly, since the optimal pH of the proteolytic enzyme used in the present invention is weakly alkaline, step (2) is carried out under a pH other than weakly alkaline, for example, under weakly acidic conditions, and the step (2) 3) is carried out under weakly alkaline conditions. “Proteolytic enzyme is active” means that the proteolytic enzyme degrades the protein. “Proteolytic enzyme is inactive” means that the proteolytic enzyme does not substantially degrade the protein.

For example, the proteolytic activity of bovine pancreatic trypsin is pH dependent and has activity at a pH of about 6-10. Based on this, by setting the pH of steps (2) to (3) to a weakly acidic condition of less than 6, and setting the pH of step (4) to a condition of about 6 to 10, steps (2) and It is possible to perform (3) under conditions where the proteolytic enzyme is inactive and to perform step (4) under conditions where the proteolytic enzyme is active.

As a more specific example, trypsin is inactive in a gel in which a solution of pH 4.7 containing trypsin and agarose is solidified. By immersing agarose in a cell lysate having a pH of 8.1, trypsin is activated and a proteolytic reaction is started.

Since the enzyme reaction is affected by temperature, enzyme concentration, reaction time, enzyme inhibitor, etc., in addition to pH, "the proteolytic enzyme is active" according to the properties of the proteolytic enzyme, using these as factors. Alternatively, the conditions of the steps (2) to (4) can be appropriately set so that “the proteolytic enzyme is inactive”.

(5) In the step (5) of separating nucleic acids derived from cells on the support treated in step (4) by pulse field electrophoresis using an electrophoresis solution containing a condensed phosphate compound, Nucleic acids derived from cells on the support treated in step (4) are separated by pulse field electrophoresis using an electrophoresis solution containing a condensed phosphate compound.

In the pulse field electrophoresis method, electrophoresis is performed by changing the direction of the electric field at a constant interval using a plurality of electrodes. Pulse-field electrophoresis induces morphological changes in nucleic acid molecules by changing the direction of the electric field at regular intervals, and moves and separates large nucleic acid molecules so that they pass through the gel network well. It is something that can be done.

In the present invention, the nucleic acid is present at the position where the cell is embedded in the gel before electrophoresis. By performing electrophoresis by pulse field electrophoresis, the nucleic acid can be extended from the origin (position where the nucleic acid exists before electrophoresis) while loosening the entangled nucleic acid in the gel.

A person skilled in the art can appropriately perform pulse field electrophoresis. For example, the electrophoresis tank used for pulse field electrophoresis is, for example, 40 to 120 degrees, more specifically 40 degrees, 45 degrees, 50 degrees, 60 degrees, 70 degrees, 90 degrees, 100 degrees, 110 degrees, and 120 degrees. Two sets of electrodes intersecting at an angle selected from the group consisting of degrees, or a plurality of three or more sets of electrodes positioned within this angle range can be provided. And the support body processed by process (4) can be set | placed on a pulse field electrophoresis apparatus so that a cell arrangement | positioning site | part may be located in the intersection, and it can electrify by electrifying alternately.

Moreover, in electrophoresis by pulse field electrophoresis, the direction of electrophoresis can be changed by rotating the support, and electrophoresis can be performed twice or more. For example, in the present invention, in the pulse field electrophoresis of step (5), after the first pulse field electrophoresis is completed, the support is rotated at a constant angle with the cell arrangement portion of the support as the center of rotation. This can be achieved by performing a second pulse field electrophoresis. In this way, by changing the direction of electrophoresis and performing electrophoresis twice or more, long nucleic acid fragments entangled in the gel that cannot be separated by the first electrophoresis can be detected.

The difference between the electrophoresis results of the one-dimensional pulse field electrophoresis and the two-dimensional pulse field electrophoresis will be described with reference to FIG. 8 schematically showing these electrophoresis results. As shown in the schematic diagram (upper diagram) of “one-dimensional electrophoresis” in FIG. 8, after one-dimensional pulse field electrophoresis, long-chain DNA extending from the origin, and fibrous DNA fragment separated from the tip (a) In addition, shorter DNA strands (b) and particulate fragments (c) separated earlier are observed. On the other hand, after two-dimensional pulse field electrophoresis, as shown in the schematic diagram (lower diagram) of “two-dimensional electrophoresis” in FIG. 8, long-chain DNA that was not elongated by the first electrophoresis is elongated, In the first dimension, a very long DNA fragment (d) that is entangled with the DNA strand extended from the origin and cannot be separated is detected. Further, the DNA fragments or particles of a, b, c, and d are migrated in different directions, and various lengths of DNA are observed in parallel in the vertical direction. Therefore, in the two-dimensional pulse field electrophoresis, if no DNA is detected in the compartment where the DNA fragments of a, b, c and d are supposed to be migrated, the intracellular DNA of the cell is not cleaved. Can be determined.

When performing two-dimensional pulse field electrophoresis, the angle at which the support is rotated after the first pulse field electrophoresis is blocked by long DNA fragments that cannot be detected by one-dimensional pulse field electrophoresis due to DNA strands extending from the origin. The angle is not particularly limited as long as the angle can be detected, but for example, 60 to 160 degrees, more specifically 60 degrees, 70 degrees, 80 degrees, 90 degrees, 100 degrees, with the cell arrangement portion of the support as the rotation center, The angle is selected from the group consisting of 110 degrees, 120 degrees, 130 degrees, 140 degrees, 150 degrees, and 160 degrees. When this angle is 140 degrees, DNA fragments / particles a, b, c and d can be clearly detected as shown in the schematic diagram on the lower side of FIG.

In the conventional COMET method, a pattern in which particulate fragments are electrophoresed from the origin is analyzed. In the conventional COMET method, the nucleoprotein is not digested using a proteolytic enzyme. Furthermore, in order to perform electrophoresis using a normal single electrode, nucleic acids entangled in the gel are released from the nucleoprotein. It could not be loosened and stretched.

The electrophoresis solution used in the step (5) is not particularly limited as long as it contains a condensed phosphate compound. For example, components of a commonly used electrophoresis buffer are added. As described in the description of the above step (4), the addition of the condensed phosphate compound can prevent reassociation of the nucleoprotein digest and DNA during electrophoresis. In previous DNA studies, DNA electrophoresis used a deproteinized preparation. However, even after treatment with proteolytic enzymes, basic peptides derived from degraded nucleoprotein are mixed in the system, so these basic peptides bind to DNA again, eliminating the negative charge of DNA. This interferes with electrophoresis. Therefore, the condensed phosphate compound is also contained in the electrophoresis solution. For the same reason, the electrophoretic solution is preferably weakly alkaline with active proteolytic enzymes in order to subject the basic peptide released from the nucleoprotein to further degradation.

The condensed phosphate compound contained in the weakly alkaline solution in step (4) and the condensed phosphate compound contained in the electrophoresis solution in step (5) are the same or different from each other. Further, each of them may be two or more kinds of condensed phosphate compounds. For example, when performing two-dimensional pulse field electrophoresis, the condensed phosphate compound contained in the weakly alkaline solution is hexametaphosphate. And the condensed phosphate compound contained in the electrophoresis solution is preferably a polyphosphate or a hexametaphosphate.

Electrophoretic conditions such as electrophoresis voltage, electrode switching timing and electrophoresis time used for pulse field electrophoresis are not particularly limited in the case of one-dimensional pulse field electrophoresis, but for two-dimensional pulse field electrophoresis, For example, the first electrophoresis is performed every 5 to 5 seconds at a potential difference of 1.0 to 2.0 V / cm for 5 to 10 minutes, and then the second electrophoresis is performed with a potential difference of 1.0 to 2 Electrophoresis is performed every 5 to 5 seconds at 0.0 V / cm for 5 to 10 minutes.

The nucleic acid extended by electrophoresis in step (5) can be stained with a nucleic acid staining dye or the like and observed with a fluorescence microscope or the like. The nucleic acid staining dye used in the present invention is not particularly limited as long as it emits fluorescence, and examples thereof include Cyber Gold. The cleavage of the nucleic acid contained in one cell is visualized and observed with a microscope or the like.

According to the present invention, double-stranded DNA constituting a chromosome is observed as a DNA fiber (for example, FIGS. 3A and B and FIG. 7). When cleavage of double-stranded DNA is started, first, several breaks occur, and several filamentous fragments are cut out (for example, FIGS. 4A and B). As the cutting progresses, the filamentous pieces to be cut out increase and shorten. As the cutting further proceeds, particulate fragments appear (eg, FIG. 4C). When the fragmentation further proceeds, the nucleic acid amount present at the origin (position where the nucleic acid exists before electrophoresis) is decreased, and is observed as a reduction of the origin under the microscope.

That is, according to the present invention, it is possible to observe and detect one to several cuts of double-stranded DNA contained in one cell, increase and shorten of cut-out filamentous fragments, and particulate fragments.

The features of the present invention are (i) performing a proteolytic enzyme treatment, (ii) not performing an alkali treatment, (iii) using a pulse field electrophoresis method, and the like.

On the other hand, in the conventional COMET method, (i) proteolytic enzyme treatment is not performed, (ii) alkali treatment is performed, and (iii) electrophoresis using a single electrode is performed. The conventional COMET method cannot obtain an appropriate result for the following reasons.

That is, (i) since no proteolytic enzyme treatment is performed, the nuclear matrix retains the DNA, resulting in false negatives (for example, FIGS. 5A and B). (Ii) When the alkali treatment is performed, the DNA is not Since it is artificially cleaved, a false positive is generated (for example, FIGS. 6A to 6D), and (iii) electrophoresis using a single electrode could not loosen and elongate the entangled nucleic acid in the gel. .

Therefore, it has been found that the conventional COMET method cannot produce an appropriate result because it produces both false negatives and false positives.

2. TECHNICAL FIELD The present invention relates to a method for producing a cell in which a nucleic acid is not cleaved. Furthermore, the specific one aspect | mode of this invention is related with the method of manufacturing the cell in which the nucleic acid is not cut | disconnected including the process of following (1)-(6). In the present invention, “producing a cell” means obtaining a specific cell. As other expressions, “producing a cell”, “isolating a cell”, “extracting a cell” Or the like.
(1) A step of placing one or a plurality of cells in a test sample on a support having an amino group on the surface (2) A weakly acidic solution comprising a purified proteolytic enzyme having a weakly alkaline optimum pH and a gelling agent The step of covering the cells with a mixture with the solution (3) The step of solidifying the weakly acidic solution (4) The support on which the weakly acidic solution is solidified comprises a condensed phosphate compound, a surfactant and a reducing agent. Step of immersing in weak alkaline solution (5) Step of separating nucleic acid derived from cells on a support immersed in the weak alkaline solution by pulse field electrophoresis using an electrophoresis solution containing a condensed phosphate compound (6) A step of determining a cell in the test sample as a cell whose nucleic acid has not been cleaved when the separation mode of the electrophoresed nucleic acid is similar to the separation mode of a nucleic acid derived from a normal cell.

The test sample is not particularly limited as long as it is a sample containing cells, and may be derived from a living body or naturally derived. For example, if it is desired to produce sperm whose nucleic acid has not been cleaved, the test sample can be used as semen.

Steps (1) to (5) can be performed in the same manner as in the method for detecting cleavage of a nucleic acid contained in one cell of the present invention. Therefore, in the present invention, step (6) will be described in detail.

(6) Step of determining a cell in the test sample as a cell in which the nucleic acid is not cleaved when the separation mode of the electrophoretic nucleic acid is similar to the separation mode of the nucleic acid derived from normal cells (6) ), When the separation mode of the nucleic acid electrophoresed in step (5) is the same as the separation mode of the nucleic acid derived from the normal cell, the cell in the test sample is regarded as a cell whose nucleic acid has not been cleaved. judge.

In the present invention, the nucleic acid separation mode refers to the mode of presence of DNA fragments / particles a, b, c and d shown in the schematic diagram (lower diagram) of the two-dimensional pulse field electrophoresis of FIG. 8, which is normal. If the degree of presence of the DNA fragments / particles a, b, c and d of the nucleic acid derived from the cell is the same, the cell in the test sample can be determined as a cell whose nucleic acid has not been cleaved. This determination may be either qualitative or quantitative. Examples of qualitative judgment and quantitative judgment are shown below. However, the determination shown below can be performed programmatically, automatically, or mechanically without depending on the determination of those skilled in the art, as is apparent from the determination criteria.

As an example of qualitative determination, the number or total number of the DNA fragments / particles a, b, c and d separated from the nucleic acid electrophoresed in step (5) and the nucleic acid derived from normal cells are separated. When the difference is not statistically significant, a cell in the test sample can be determined as a cell whose nucleic acid has not been cleaved.

As an example of quantitative determination, the number or total number of DNA fragments / particles a, b, c, and d separated from the nucleic acid electrophoresed in step (5) and normal cells are used as shown below. The possibility (%) that the cells in the test sample are not cleaved nucleic acid is set in advance for each range of correlation with those separated from the nucleic acid, and the cells in the test sample and normal cells The possibility (%) is determined from the nucleic acid separation mode: when the correlation is A to B, the possibility that the cell in the test sample is determined as a cell whose nucleic acid has not been cleaved is 10% or less; If the correlation is B to C, the probability of determining a cell in the test sample as a cell that has not been cleaved by the nucleic acid is 10% to 30%; if the correlation is C to D, The cells as non-nucleic acid-cleaved cells The ability is 30% to 50%; if the correlation is D to E, the probability of determining a cell in the test sample as a cell that has not been cleaved nucleic acid is 50% to 70%; Is E to F, the probability of determining cells in the test sample as cells that have not been cleaved nucleic acid is 70% to 90%; if the correlation is greater than F, the cells in the test sample are Is determined to be an uncut cell.

However, in normal cells, there is a probability that the DNA fragments / particles a, b, c and d cannot be detected even when subjected to electrophoresis in the step (5). Therefore, in step (6), the separation mode of nucleic acids derived from normal cells can be set as a separation mode in which the number of DNA fragments / particles a, b, c and / or d is zero.

A normal cell refers to a cell in which no abnormality is observed in the characteristics of the cell such as a phenotype such as morphology and proliferative property and a genotype such as expression of a specific gene. Further, the normal cell may simply be a control cell (control cell) set by the subject.

3. The kit of the present invention The present invention relates to a kit for carrying out the method of the present invention. Furthermore, one specific aspect of the present invention relates to a kit for detecting nucleic acid cleavage or producing a cell in which nucleic acid is not cleaved, which comprises the following components (1) to (5).
(1) Support having an amino group on the surface (2) Purified proteolytic enzyme having an optimum pH weakly alkaline (3) Weakly acidic solution containing a gelling agent (4) Condensed phosphate compound, surfactant and reduction Weak alkaline solution containing an agent (5) Electrophoretic solution containing a condensed phosphate compound

The components (1) to (5) contained in the kit of the present invention are those described in the method of the present invention. The kit of the present invention can further contain the following component (6).
(6) Electrophoresis device having two or more sets of electrodes, and the two or more sets of electrodes intersecting at an angle of 45 to 120 degrees

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to an Example.

1. Purified Trypsin Commercially available bovine pancreatic trypsin preparation usually has an active trypsin content of 20% or less due to autolysis and the like. Moreover, since the commercially available bovine pancreatic trypsin preparation is derived from the pancreas, a DNA-degrading enzyme is mixed as an impurity. Trypsin was purified by affinity chromatography with immobilized Lima bean trypsin inhibitor (LBTI). The column packed with the LBTI-immobilized adsorbent was previously equilibrated with 20 mM Tris-HCL (pH 8.3) and 1.0 mM CaCl ₂ . Trypsin powder was dissolved in the same solution and added to the column. Using the equilibrated solution to which 0.2 M NaCl was added, non-adsorbing impurities were washed out with a spectrophotometer until the absorption at 280 nm of the eluate was 0.01 or less. Adsorbed trypsin was eluted with a 2.0% acetic acid solution (pH 1.8). The obtained preparation was diluted with a 2.0% acetic acid solution to make a trypsin concentration of 200 μg / ml, and then stored frozen.

2. Pulse field electrophoresis (1)
(1) Method To purify motile sperm from human semen, the Percoll density gradient centrifugation method and the swim up method were combined. Using a cytospin apparatus (Sakura Seiki), purified motor sperm of 2 × 10 ⁴ cells were smeared on a slide glass in a range of 7 × 7 mm. The amino group was labeled on the surface of the slide glass.

Dissolved 0.56% agarose (0.1 M sodium acetate (pH 4.7), 0.05% Triton X-100) was filtered through a 0.22 μm Millipore filter, and 540 μl thereof and 60 μl of purified trypsin were mixed at 40 ° C. did. The prepared agarose was embedded to a thickness of 50 μm in the range of 2.5 × 2.5 cm around the sperm smearing position, and solidified at low temperature for 30 minutes. The solidified gel was immersed in a cell lysis solution (30 mM Tris-polyphosphoric acid, 8.2 mM Na hexametaphosphoric acid, 0.05% Triton X-100, 5.0 mM dithiothreitol, pH 8.1) at 37 ° C., The reaction was allowed for 30 minutes.

The electrophoresis tank used for pulse field electrophoresis was one having two sets of electrodes intersecting at an angle of 90 degrees. As the electrophoresis solution, 30 mM Tris-polyphosphoric acid (pH 8.1) was used. A slide glass was placed so that the cell smear portion was located at the intersection of the two sets of electrodes, and the electrophoresis was performed for 7 minutes by alternately energizing every 3 seconds at a potential difference of 1.5 V / cm.

(2) Results When purified motor sperm was suspended in a cell lysate added with SyberGold without digesting cells with proteolytic enzymes, the sperm head swelled in a few minutes, but the DNA fiber swelled. It remained in the sperm head (FIG. 1A). On the other hand, when purified trypsin (20 μg / ml) was added to the suspension, DNA fibers were observed to diffuse around the head in several tens of seconds (FIG. 1B). Thus, in the suspension, the DNA fiber is free-diffusing, so that it is difficult to specify the DNA fiber derived from one cell and its fragment.

Therefore, when the purified motile sperm and ejaculated semen were embedded in agarose and trypsin was added to the cell solution, the purified motile sperm showed a state in which the ends of the entangled DNA fibers protruded to the periphery (Fig. 2A). On the other hand, various forms were observed in ejaculate, and in addition to the form seen in purified motile sperm, what showed the appearance of particulate fragments concentrically diffusing around was observed (FIG. 2B). These are free diffusion in the agarose network structure without applying a voltage. From the particulate fragments found in ejaculates, the sperm in the semen is likely to be sperm that has already undergone DNA fragmentation.

Subsequently, sperm that had been lysed in the presence of trypsin was electrophoresed by pulse field electrophoresis. In purified motile spermatozoa, dozens of uniform DNA fibers were elongated from the origin (position where the nucleic acid was present before electrophoresis) (FIG. 2C). On the other hand, very various electrophoretic images were observed in ejaculate (FIG. 2D). Among ejaculates, in addition to the elongated DNA fibers observed when purified motor spermatozoa were used, there were those in which filamentous fragments separated from the tips of the elongated DNA fibers were observed. The number, length, etc. of the filamentous pieces varied greatly depending on the sperm.

FIG. 3A is a photograph showing an enlarged view of a DNA fiber stretched by pulse field electrophoresis, and FIG. 3B is a photograph showing an enlarged view of the tip portion of the DNA fiber.

When the electrophoresed DNA fiber was examined at a high magnification, it was observed that the DNA fiber extended in a zigzag manner in the agarose network structure by applying a voltage by pulse field electrophoresis.

Purified motile spermatozoa are divided into a group in which only DNA fibers are observed (FIGS. 3A and B), and a group in which several filamentous fragments are observed at the tips of the DNA fibers (FIGS. 4A to 4C). It was. That is, the present inventor has found a surprising result that even purified sperm spermatozoa exist at an early stage of fragmentation. The present invention has extremely excellent sensitivity capable of detecting even one to several cleavages of DNA contained in one cell, and can detect an initial stage of DNA cleavage.

The present inventor further conducted the following experiment in order to show that even purified motile spermatozoa exist in the initial stage of fragmentation.

To examine the number of purified motile sperm fragments, as described above, semen (n = 8; vol = 2.5 ± 0.62 ml; concentration = 58 ± 26 × 10 ⁶ sperm / ml; motility rate = 59 ± 17%) to obtain purified motor sperm (vol = 1.0 ml; concentration = 5.3 ± 2.7 × 10 ⁶ sperm / ml; motility = 94 ± 2.1%) It was. Subsequently, the obtained purified motor sperm was subjected to electrophoresis by pulse field electrophoresis according to the method described above. The percentage of purified motile spermatozoa in which no fragment was detected was 58.9% to 87.2% (77.7 ± 9.47%). In all specimens, the rate of purified motile spermatozoa with no fragments detected was significantly lower compared to the motility rate. This indicates that even purified motile spermatozoa are present in the early stages of fragmentation.

(3) Comparative Example Hereinafter, a comparative example will be shown in order to prove the above-mentioned “comparison between the present invention and the conventional COMET method”.

(I) Comparative Example when Trypsin is not Used When cell lysis is performed without adding trypsin, the electrophoretic image in the pulse field is completely different from that in which cell lysis was performed by adding trypsin. became. That is, in the case of purified motile sperm, the DNA fiber did not extend from the origin (position where the nucleic acid was present before electrophoresis) (FIG. 5A). In addition, in the case of ejaculated semen, DNA fibers could not be observed only by migration of a small amount of particulate fragments (FIG. 5B). This indicates that when trypsin is not added, the DNA is retained in the nuclear matrix, so that it is impossible to observe the DNA fiber and the filamentous fragments cut out therefrom by electrophoresis. .

(Ii) The effect of alkali treatment on the comparative DNA in the case of alkali treatment was observed. Purified motile spermatozoa that had been lysed with trypsin were immersed in 0.05 or 0.1 mol / L NaOH solution at room temperature for 10 minutes, washed with water, and subjected to pulse field electrophoresis. In the case of 0.05 mol / L NaOH solution, only DNA particulate fragments were migrated and no DNA fiber was observed (FIG. 6A). In the case of a 0.1 mol / L NaOH solution, the nucleic acid at the origin (position where the nucleic acid was present before electrophoresis) was reduced, and fragmentation further proceeded (FIG. 6B).

Next, when the purified motile sperm was immersed in a 0.1 mol / L NaOH solution after pulse field electrophoresis, the elongated DNA fibers were completely fragmented (FIG. 6C). On the other hand, only a small amount of particulate fragments migrated in purified motor sperm that had been lysed with 0.1 mol / L NaOH solution without adding trypsin (FIG. 6D).

Therefore, in the COMET method in which alkali treatment is performed, there is a high possibility of false positives for DNA fragmentation, whereas in the present invention in which alkali treatment is not performed, DNA is not cleaved by alkali treatment, Expect no positive results.

(4) Regarding the cells other than sperm The above results are obtained when sperm is used, but for the following reasons, in the cells other than sperm, as in sperm, the nucleic acid contained in one cell is cleaved. Can be detected.

The present invention is characterized in that cell nuclear DNA can be separated from nuclear proteins and nuclear matrix by adding a proteolytic enzyme into the gel and subjecting it to proteolytic enzyme treatment. In cells other than sperm, intracellular DNA is immobilized on nuclear proteins and / or nuclear matrix. Therefore, by using the present invention, it is possible to detect the cleavage of a nucleic acid contained in one cell even in cells other than sperm, similarly to sperm.
3. Pulsed field electrophoresis (2)
(1) Method To purify motile sperm from human semen, the Percoll density gradient centrifugation method and the swim up method were combined. Using a cytospin apparatus (Sakura Seiki), purified motor sperm of 2 × 10 ⁴ cells were smeared on a slide glass in a range of 7 × 7 mm. The amino group was labeled on the surface of the slide glass.

Dissolved 0.3% agarose (0.1 M sodium acetate (pH 4.7), 0.05% Triton X-100) was filtered through a 0.22 μm Millipore filter, and 540 μl thereof and 60 μl of purified trypsin were mixed at 40 ° C. did. The prepared agarose was embedded to a thickness of 50 μm in the range of 2.5 × 2.5 cm around the sperm smearing position, and solidified at low temperature for 30 minutes. The solidified gel was immersed in a cell lysis solution (30 mM Tris-hydrochloric acid, 1.0 mM Na hexametaphosphoric acid, 0.05% Triton X-100, 5.0 mM dithiothreitol, pH 8.1), 37 ° C., 30 Reacted for 1 minute.

The electrophoresis tank used for pulse field electrophoresis was one having two sets of electrodes intersecting at an angle of 60 degrees. As the electrophoresis solution, 30 mM Tris-acetic acid, 1.0 mM Na-hexametaphosphoric acid (pH 8.1) or a solution containing 1.0 mM Tris-acetic acid and 30 mM Tris-polyphosphoric acid (pH 8.1) was used. A slide glass was placed so that the cell smear portion was located at the intersection of the two sets of electrodes, and the electrophoresis was performed for 7 minutes by alternately energizing every 3 seconds at a potential difference of 1.5 V / cm. Next, the slide glass was rotated 140 degrees with the cell arrangement portion of the support as the center of rotation, and was similarly subjected to electrophoresis for 7 minutes. Cybergold, a DNA fluorescent staining reagent, was dropped on the cell smear site on the slide glass after the electrophoresis to stain the DNA. The stained DNA was observed with a fluorescence microscope.

(2) Results FIG. 7 shows the results of microscopic observation of DNA subjected to two-dimensional pulse field electrophoresis according to the method described above. The arrows in the figure indicate long DNA fragments that cannot be observed by one-dimensional pulse field electrophoresis. By observing these long-chain DNA fragments, nucleic acid separation by this two-dimensional pulse field electrophoresis can detect cells with fragmented DNA in the cell nucleus with higher sensitivity compared to nucleic acid separation by one-dimensional pulse field electrophoresis. I found out that I can do it. In particular, since DNA fragmentation occurs at random, if long-chain DNA fragments cannot be observed, it cannot be confirmed whether or not DNA in the cell nucleus is fragmented. That is, as shown in the lower diagram of FIG. 8 schematically showing DNA fragmentation, as one embodiment of DNA fragmentation, long-chain DNA fragmentation (d in the figure) occurs, and then, by long-chain DNA fragmentation, It is conceivable that short-chain DNA fragmentation (same as a and b) occurs, and finally DNA particleization (same as c) occurs. In this embodiment, even if the short-chain DNA fragment (a, b) or the DNA particle (c) cannot be detected by the one-dimensional pulse field electrophoresis, the long-chain DNA fragment (d) cannot be detected. For example, cells in the initial stage of DNA fragmentation cannot be observed. Nucleic acid separation by this two-dimensional pulse field electrophoresis solves this problem of nucleic acid separation by one-dimensional pulse field electrophoresis, and long-chain DNA that could not be observed by conventional one-dimensional pulse field electrophoresis. By making it possible to observe the fragment (d), cells in the initial stage of DNA fragmentation can be observed with high sensitivity.

Claims (7)

A method for detecting cleavage of a nucleic acid contained in one cell, comprising the following steps (1) to (5).
(1) Step of placing one or a plurality of cells on a support having an amino group on the surface (2) A mixture of a purified proteolytic enzyme having an optimum pH weakly alkaline and a weakly acidic solution containing a gelling agent (3) solidifying the weak acid solution (4) solidifying the weak acid solution into a weak alkaline solution containing a condensed phosphate compound, a surfactant and a reducing agent. (5) A step of separating nucleic acids derived from cells on a support immersed in the weak alkaline solution by two-dimensional pulse field electrophoresis using an electrophoresis solution containing a condensed phosphate compound. The two-dimensional pulse field electrophoresis uses an electrophoresis apparatus having two or more sets of electrodes and the two or more sets of electrodes intersecting at an angle of 45 to 120 degrees. , After the first pulse field electrophoresis, the support, as the center of rotation of the cell arrangement portion, is achieved by performing a second pulse field electrophoresis is rotated 60 to 160 degrees, said step A method for producing a cell in which a nucleic acid is not cleaved, comprising the following steps (1) to (6).
(1) A step of placing one or a plurality of cells in a test sample on a support having an amino group on the surface (2) A weakly acidic solution comprising a purified proteolytic enzyme having a weakly alkaline optimum pH and a gelling agent The step of covering the cells with a mixture with the solution (3) The step of solidifying the weakly acidic solution (4) The support on which the weakly acidic solution is solidified comprises a condensed phosphate compound, a surfactant and a reducing agent. Step of immersing in weak alkaline solution (5) Separation of nucleic acids derived from cells on a support immersed in the weak alkaline solution by two-dimensional pulse field electrophoresis using an electrophoresis solution containing a condensed phosphate compound The two-dimensional pulse field electrophoresis has two or more sets of electrodes, and the two or more sets of electrodes intersect at an angle of 45 to 120 degrees. Dress After performing the first pulse field electrophoresis using, the substrate is rotated by 60 to 160 degrees with the cell arrangement portion as the rotation center, and the second pulse field electrophoresis is performed. Step (6) When the mode of separation of the electrophoresed nucleic acid is similar to the mode of separation of a nucleic acid derived from normal cells, the cell in the test sample is determined as a cell whose nucleic acid has not been cleaved. Process The purified proteolytic enzyme having an optimum pH that is weakly alkaline has an activity of decomposing a peptide bond between arginine and any amino acid and / or a peptide bond between lysine and any amino acid. the method of. The method according to claim 1 or 2, wherein the purified proteolytic enzyme having an optimum pH that is weakly alkaline is a purified trypsin-like enzyme. The method according to claim 1 or 2, wherein the condensed phosphoric acid compound is at least one condensed phosphoric compound selected from the group consisting of polyphosphoric acid, hexametaphosphoric acid and salts thereof. The method according to claim 1 or 2, wherein the cell is a sperm. The kit for using in the method of Claim 1 or 2 containing the component of following (1)-(6).
(1) Support having an amino group on the surface (2) Purified proteolytic enzyme having an optimum pH weakly alkaline (3) Weakly acidic solution containing a gelling agent (4) Condensed phosphate compound, surfactant and reduction (5) Electrophoretic solution containing condensed phosphate compound (6) Two or more electrodes, and the two or more electrodes are 45 to 120 degrees Electrophoresis device that crosses at an angle

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