JP2003066003A - Simple filling method for polymer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device for micro-channel electrophoresis, which has improved separative ability and analytic accuracy. SOLUTION: In this device for electrophoresis of a microchip, a water soluble high polymer is filled in a micro-channel on the microchip as migration liquid with concentration graded.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a device for microchip electrophoresis. More specifically, the electrophoretic liquid is filled in the microchannels (hereinafter referred to as microchannels) of the electrophoretic microchip, and the water-soluble polymer is a component of the electrophoretic liquid. The present invention relates to a device for microchip electrophoresis, wherein the ionic polymer has a stepwise concentration gradient.

[0002]

2. Description of the Related Art Capillary electrophoresis (Capillary Elec
trophoresis (hereinafter abbreviated as CE) is widely used for separating nucleic acids and includes polymerase chain reaction (P
It has been shown to be effective in the analysis of (CR) products, separation of restriction enzyme fragments, DNA sequencing, etc. (eg Cohen, AS; Najarian, D .; Smith, J, A .;
Karger, BLJChromatogr.1988,458,323-333, Kheterpa
l, I.; Mathies, RAAnal.Chem.1999,31A-37A, Heller, C.
Electrophoresis, 2001, 22, 629-643). for that reason,
Capillary electrophoresis is based on the Human Genome Project (Human Geno
me Project; HGP). However, in recent years, it has become possible to reduce the size of analytical instruments due to the development of microelement manufacturing technology and the electronics industry. Therefore, in place of glass capillaries that are cumbersome to handle, high-speed analysis, downsizing of devices, chemicals and DJ Harrison et al./Science 261 (199) is a form that can be expected to reduce sample consumption.
3) As shown in 895-897 and Anal.Chim.Acta283 (1993) 361-366, a chip (particularly a microchip) formed by joining two substrates has been proposed.

In conventional microchip electrophoresis, DNA samples are generally separated by one type of matrix at a fixed concentration. Therefore, when the sample DNA is a complex DNA composed of various bp (base pair) fragments, the separability between the small fragment and the large fragment is not always satisfactory, and therefore, the small fragment and the large fragment are simultaneously separated. It was difficult to separate and collect. The same problem is found in CE, and in order to solve it, for example, a method of mixing several polymers in a buffer solution (eg, Liang, DH; Chu, B. Electrophoresis, 1
998,19,2447-2453, Liang, DH; Song, LG; Zhou, SQ; Z
aitsev, VS; Chu, B.Macromolecules, 1999,20,6326-6332
Etc.) and methods using electric field gradients (eg Luckey, JA; Sm
ith, LMAnal.Chem.1993,65,2841-2850, Endo, Y.; Yoshi
da, C.; Baba, YJBiochem.Biophys.Methods 1999,41,133
-141 etc.) has been proposed. However, the stepwise concentration gradient of the polymer solution in CE is due to the electroosmotic flow
It was possible only in the presence of oosmotic flow (EOF) (eg Chen HS.; Chang HT.J.Chromatogr.A, 1
999,853,337-347, Chen HS.; Chang HT.J.Chromatogr.
A, 1999,853,337-347). When the inner surface of the capillary is negatively charged, positively charged ions and particles gather near the inner wall surface of the capillary, so when an electric field is applied, the ions and particles flow toward the negative electrode side near the inner surface of the capillary. Will end up. EOF is the reverse flow that occurs in the center of the capillary to correct it.

The speed at which the sample is carried by the EOF is about several μm / millisecond, but the sample area (also called the sample plug) is delayed due to the delay in switching the high voltage.
Moves from several μm to several tens of μm. Since the size of the sample plug is generally several tens of μm, there is a problem in that unfavorable phenomena such as spread of the sample plug and deterioration of analysis accuracy are induced due to movement of the sample plug due to delay in switching high voltage. there were.

[0005]

In view of the above problems, the present invention provides a stepwise concentration gradient of a water-soluble polymer matrix that can be easily formed on a microchip for electrophoresis.
The aim is to improve the resolution and analytical accuracy of the sample in the absence of OF.

[0006]

Means for Solving the Problems As a result of intensive studies by the present inventors, microchip electrophoresis characterized in that a water-soluble polymer is filled in a microchannel on a microchip with a concentration gradient. We have succeeded in manufacturing the device, and have found that the use of the device improves the resolution of the sample and the detection accuracy even without the presence of EOF. As a result of further studies, we have completed the present invention. It was

That is, the present invention provides (1) an apparatus for microchip electrophoresis, wherein a water-soluble polymer is filled as a running solution in a microchannel on a microchip with a concentration gradient, ( 2) The device for microchip electrophoresis according to the above item (1), wherein the electrophoretic solution further contains a buffer solution, and (3) the water-soluble polymer is a non-crosslinked water-soluble polymer. Any of the above-mentioned (1) to (3), characterized in that the device for microchip electrophoresis according to the above (1) or (2), (4) the number of types of non-crosslinking water-soluble polymer is two or more. (5) The non-crosslinked water-soluble polymer is hydroxymethyl cellulose and / or methyl cellulose, described in any one of (1) to (4) above. Microchip electrophoresis apparatus, (6) Concentration of hydroxymethyl cellulose for the entire electrophoresis solution is 0.01 to 2.0% (w /
w), the concentration of hydroxymethyl cellulose is 0.2 to
2.0% (w / w) and the volume ratio of both is 0.1 to 0.1%.
10: 0.1-10, wherein the above (1)
To (5), the microchip electrophoresis apparatus according to any one of (5) to (7), wherein the concentration of methylcellulose is 0.01 to 0.5% (w / w), and the concentration of methylcellulose is 0. 6 to 2.0% (w / w), and the volume ratio of the both is 0.1 to 5: 0.2 to 10. Any of the above (1) to (6) (8) The concentration of methylcellulose in the whole electrophoretic solution is 0.01 to 2.0% (w /
w), the concentration of hydroxymethyl cellulose is 0.01 to
0.60, and the concentration of hydroxymethyl cellulose is 0.61 to 2.0% (w / w), and the volume ratio of the three is 0.
1-5: 0.1-10: 0.1-10 The microchip electrophoresis apparatus according to any one of (1) to (7) above, characterized in that:

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Microchip electrophoresis has a width and depth of, for example, several to 100 .mu.m formed on a microchip widely used in the field of electronics.
Especially by performing electrophoresis in a groove of about m
This is an effective method for separating A. Since the length of the separation channel used for separation is often several cm, the migration time is usually short (several seconds to several tens of seconds). The microchip has a high degree of freedom in circuit design, and since it is easy to form a multi-capillary by arranging a plurality of channels on the same chip, it has an advantage that the processing capacity can be easily increased.

The microchip electrophoresis apparatus in the present invention may be one known per se. A commercially available device can also be used, and examples thereof include an SV1100 microchip electrophoresis device with a HITACHI LED detector (manufactured by Hitachi Electronics Engineering Co., Ltd.). More specifically, one mode of the apparatus will be described below with reference to FIG. The electrophoretic microchip 1 is composed of a pair of substrates 2 and 3 made of a glass plate, a quartz plate, or the like (FIG. 1A). The sample introduction flow path 4 and the separation flow path 5 are formed on the upper surface of the lower substrate 2 so as to intersect with each other (referred to as “intersection 7”) (FIG. 1 (C)),
Through holes 6 are formed in the upper substrate (hereinafter abbreviated as a cover body) 3 at positions corresponding to the respective ends of the grooves 4 and 5 (FIG. 1 (B)). The grooves 4 and 5 are formed on the surface of the substrate 2 by etching.

In the electrophoretic microchip 1, the above-mentioned pair of substrates 2 and 3 are attached to each other with the grooves 4 and 5 inside so that the first and second electrodes are formed by the through holes 6.
A sample introduction channel 4 that communicates with the outside through the reservoirs R1 and R2, and a separation channel 5 that communicates with the outside through the third and fourth reservoirs R3 and R4 are formed inside. As shown in FIG. 1, each of the reservoirs R1 to R4 is a capillary (capillary tube) for introducing a liquid such as an electrophoretic liquid from the outside.
And an electrode (not shown) for applying a voltage to the electrophoretic solution stored therein.
Are provided respectively.

The electrophoretic microchip used in the present invention includes, for example, laser processing, ion etching processing,
Approximately 10-100 by using optical lithography
It is composed of a pair of substrates made of, for example, glass or acrylic, in which microchannels having a width of about μm and a depth of about 5 to 50 μm are formed. It is considered that the above-mentioned substrate can be processed to form microchannels, has excellent chemical resistance, and also has appropriate rigidity. For example, glass, quartz, ceramics, silicon or metal, PDMS. (Polydimethylsiloxane),
It may be a material such as a polymeric material such as PMMA (polymethylmethacrylate). The same applies to the cover body. However, when considering the application of optical analysis,
It is preferably highly transparent.

The substrate may be a single or multilayer stack. Further, the substrate and the cover body are joined by heat treatment,
Alternatively, they can be laminated and integrated by means such as adhesion. In addition, at the time of this lamination, a support may be laminated and integrated on the side opposite to the cover for reinforcement of the substrates.

When the sample injection small hole, the discharge small hole, and further the agent injection small hole are arranged in the cover body, it is exemplified that the diameter thereof is several mm or less. Here, it is noted that the agent may be various ones, for example, a solvent for solvent extraction, a reaction reagent,
Alternatively, it is a diluent or the like. The processing of these small holes can be performed chemically, mechanically, or by various means such as laser processing and ion etching processing.

The microchannels can be formed by means such as laser processing and ion etching processing. The length of the microchannel is not particularly limited and can be arbitrarily set as necessary, but is preferably set to a length of about 15 to 30 mm. The width and depth of the microchannel are not particularly limited, but preferably the width is about 10 to 100 μm and the depth is about 5 to 10 μm. The shape of the microchannel may be, for example, a cross shape, but other shapes may be used.

The microchannel is in a state of communicating with the outside world only through small holes provided in the cover body such as small holes for sample injection. Therefore, the fluid flowing in the microchannel is less affected by external conditions, and becomes a more stable fine flow. Liquid can be passed through the microchannel through a conduit such as a capillary or a rubber tube. Liquid passing through a conduit such as a capillary or a rubber tube is because one end of the conduit is inserted into a sample injection small hole, a discharge small hole, and further an agent injection small hole.
For the solution, attach a microsyringe to the other end of the conduit,
This can be done by pressing the syringe. In addition, it is possible to use mechanical means such as a micropump, electric shaking means, or suction by a small hole for discharge.

In the present invention, a method of filling the separation channel with an electrophoretic solution containing a water-soluble polymer and performing electrophoresis is preferable. The electrophoretic solution according to the present invention contains a water-soluble polymer and a buffer solution, and thus has a network structure due to the entanglement of the macromolecules in the electrophoretic solution and an interaction between the water-soluble polymer and DNA to give a specific high-order The structured DNA can be separated. By using this method, separation with different selectivity can be performed only by changing the kind or concentration of the water-soluble polymer.

As the water-soluble polymer, non-crosslinked water-soluble polymer is preferably used, and specifically, for example, agarose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose (hereinafter abbreviated as HPMC), hydroxy cellulose, methyl cellulose (hereinafter , Abbreviated as MC), polysaccharides such as pullulan and dextran, polyethylene oxide, polyethylene glycol, polypropylene glycol, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone,
Examples thereof include polyols such as polyglycerin.
Preferably, hydroxypropylmethyl cellulose (H
PMC) and methyl cellulose (MC).

Examples of the buffer to be mixed with the water-soluble polymer include Tris buffer, phthalate buffer, boric acid
A Tris buffer or the like can be used, and a commercially available one may be used. Examples of such commercially available products include boric acid-Tris buffer (manufactured by Kanto Chemical Co., Inc.) and the like. As a method for adjusting a running solution by mixing a water-soluble polymer and a buffer solution, two different water-soluble polymers having different concentrations are used.
It is desirable to fill one or more kinds prepared and mixed with a buffer solution, preferably one by one, in a uniform volume ratio. The electrophoresis solution may be filled in order from the highest concentration, or from the lowest concentration. The higher the concentration of the water-soluble polymer, the higher the viscosity of the mixture of the buffer solution and the water-soluble polymer, and the viscosity causes resistance, so that a stepwise gradient is formed in the microchannel. The electrophoresis solution with a high concentration of water-soluble polymer and the electrophoresis solution with a lower concentration of water-soluble polymer that are filled afterwards contact with each other with a boundary inside the microchannel, but the diameter of the microchannel is extremely small. Because
There is no chance that both parties will spread and mix. The boundaries of the different running solutions are visible.

The running solution containing the above water-soluble polymer is
Other ingredients known in the art may be included. Examples of such other components include components that intercalate with DNA. As a result, the resolution of electrophoresis is improved, so that it is preferable to include the component in the present invention. The component that intercalates with DNA usually means a planar molecule that is inserted into double-stranded DNA such as acridine dye, but in the present invention, it is inserted into a single strand having a higher-order structure. It is a concept that also includes molecules. Examples of components that intercalate into DNA include ethidium bromide (hereinafter sometimes abbreviated as EtBr) and YO-PRO-
1, SYBR Green and the like. In the present invention, EtBr is preferably about 0.3 to 3.0 μg.
/ Ml, preferably about 0.5 to 1.0 μg / ml.

By using a conduit such as the above-mentioned capillary, an electrophoretic solution is introduced into the microchannel. The water-soluble polymer-containing electrophoretic solution is preferably introduced into the microchannel in order of increasing viscosity. Examples of the conduit include silica capillary tubes and rubber capillary tubes having an inner diameter of about 100 μm, and the capillaries included in commercially available electrophoresis chip kits may be used.

The sample that can be used in the electrophoresis of the present invention includes, for example, nucleic acids including DNA fragments of various sizes, organic molecules such as amino acids, peptides and proteins, metal ions and the like. In addition, a part of the gene is amplified by the PCR method, and the obtained double-stranded DNA (hereinafter, referred to as PCR product) is subjected to heat treatment or the like to form single-stranded DN.
Those dissociated into A can also be used. It is preferable to use the DNA diluted with, for example, Dnase-free water or Rnase-free water.

The method of injecting the sample into the sample small hole is not particularly limited, and specific examples thereof include an injection method using a syringe, a pressurization method and an electrophoresis method. The method using a syringe is the same as the method of filling the running solution into the microchannel. The pressurizing method is a method of injecting a sample by applying a pressure to the sample storage tank R4 with nitrogen gas or the like. Electrophoresis is a method of injecting a sample by applying a short high-piezoelectric pulse across a microchannel. In the present invention, the method using a syringe is preferable.

In order to observe the electrophoretic migration pattern, the PCR product or the like is labeled with a fluorescent substance or the like,
There are a method of observing the labeling substance as a mark and a method of observing the electrophoresis pattern without labeling the PCR product. The former has the advantage that it can be detected with high sensitivity. On the other hand, the latter has the advantage that it is not necessary to perform PCR twice for labeling. In particular, the method for measuring the ultraviolet absorption of DNA is the simplest and quickest method without the need to stain the migration pattern after electrophoresis. The dyeing method may be a method known per se, and specific examples thereof include ethidium bromide dyeing. Staining with ethidium bromide may be carried out according to a method known per se (Manitis
1982 Ed., Molecular Clonin
g: A Laboratory Manual, New
York, Cold Spring Harbor
Laboratory).

The conditions for electrophoresis may be according to the conditions known per se. For example, after injecting the sample into the sample injection small holes, electrodes are respectively inserted, or the electrodes previously formed in the respective reservoirs are used to prepare the sample. A high voltage of about 300 V is applied to one end of the introduction channel for a predetermined time, and the other end is GN
Bring the sample to the intersection by applying a D voltage (ground). Next, when a voltage of about 400 to 800 V, more preferably about 500 to 750 is applied to one end of the separation channel and the other end is grounded, the sample present at the intersection is introduced into the separation channel due to the potential difference. Separated by running. The electrophoresis time cannot be generally stated because it depends on the base sequence of the sample to be used or the temperature, but it is about several seconds to several tens of seconds. In order to prevent the diffusion of the sample at the intersection, a weak voltage may be applied to the separation channel when the sample is introduced to the intersection and to the sample introduction channel when the sample is separated.

The detection of the sample is carried out at the detection position. For example, the photomultiplier tube receives the luminescence generated by the contact between the sample component and the luminescent reagent, and the electric signal corresponding to the luminescence intensity can be taken out from the photomultiplier tube. The method of measuring by this is mentioned. Alternatively, a UV on-column detector, a diode array detector, a photodiode detector, or a photodiode array detector measuring its absorption spectrum can be used.

More specifically, the above microchip electrophoresis apparatus is used, for example, in the following procedure.
Both substrates 2 and 3 of the microchip electrophoretic device are overlapped, and a migration liquid composed of a buffer solution and a water-soluble polymer is introduced from one of the reservoirs (a plurality of reservoirs may be provided), and a liquid is introduced from the outside installed in each reservoir. Inject by using, for example, a vaccination syringe from the other end of the capillary for
The sample introduction flow path 4 and the separation flow path 5 are filled with an electrophoretic solution containing a water-soluble polymer. Next, while injecting a predetermined amount of a small amount of sample into the first reservoir (sample injection point) R1, the third and fourth reservoirs (agent injection point) R3, R
4. An electrophoretic solution (for example, 0.1 M Tris-borate buffer) is injected into No. 4. The sample and the running solution are injected in the same manner as the method of filling the running solution.

After injecting the sample, the electrophoretic solution and the water-soluble polymer as described above, an electrode is inserted into each reservoir, or one end of the sample introduction channel 4 is formed by using an electrode previously formed in each reservoir. Apply a predetermined high voltage for a predetermined time (for example, 300V for 60 seconds), and apply the other end to G
Set to ND potential (ground). Further, a low voltage is applied to both ends of the separation channel 5 to suppress the diffusion of the sample into the separation channel (hereinafter, in the separation channel, the base end side in the migration direction is one end and the end portion in the traveling direction is the other end. The end). As a result, the sample moves in the sample introduction flow path 4, and after a predetermined voltage application time elapses,
Guided to the intersection 7.

Next, one end of the separation channel 5 is connected to about 550-75.
A voltage of about 0 V is applied, the other end is switched to the GND potential, and the sample is separated by the difference in charge, size, and the like. At this time, for example, about 130
By applying a voltage of about V, the diffusion of the sample into the separation channel is suppressed.

The sample is separated according to the above procedure to form the separation channel 5.
After the separation is performed in step 1, the detection is performed by the detector at the detection position on the other end side of the separation channel 5. For example, when the sample is migrated in the separation channel 5, the components contained in the sample are separated and arrive at the fourth reservoir R4 with a time lag. In contact with the luminescent sample input from the third reservoir, a chemical reaction occurs in the separation channel 5 to emit light. There is a method in which the generated light emission is received by a photomultiplier tube and an electric signal corresponding to the intensity of the emitted light can be taken out from the photomultiplier tube for measurement. In addition, about 200 ~
It is also possible to use a photodiode detector that observes the change in absorbance at a wavelength of 300 nm, or a photodiode array detector that measures the absorption spectrum. Also, D
When NA is fluorescently labeled, fluorescence emission can be detected. This enables highly sensitive detection.
Fluorescence emission detection is preferably performed at an excitation wavelength of about 240 to 600 nm, although it depends on the fluorescent substance used. The detector is linked to an integrator for recording electrophoretic peaks.

[0030]

[Test Example 1] Effect of MC concentration on separation of 20 bp DNA As a sample, 20 bp DNA (GenSur (GenSur
a) Purchased from Laboratories) 2 μg / ml DNAase-
It was diluted with free water (ICN Biomedicals). 0.1% (w
/ W), 0.3% (w / w), 0.5% (w / w) and 0.7% (w / w) MC (viscosity at 20 ° C. 2
% Aqueous solution, Sigma Chemical Co., Ltd.) 50 mM each
100% of boric acid-Tris buffer (manufactured by Kanto Chemical Co., Inc.)
The solution was added at 0 ° C. and stirred at room temperature until the solution became uniform and transparent to obtain four kinds of solutions. 0.5 μg / ml EtBr (manufactured by Nippon Gene Co., Ltd.) was added to each of the obtained solutions to prepare four kinds of electrophoretic solutions.

The electrophoresis was carried out using an SV1100 microchip electrophoresis apparatus (manufactured by Hitachi Electrox Engineering) equipped with a HITACHI LED detector.
The four kinds of electrophoresis solutions prepared by the above method were mixed with HITAC.
It was introduced into the microchannel on the chip using the tube of the HI electrophoresis chip kit. Sample is 300
It was applied with V for 60 seconds. The separation voltage was set to 130 V for the sample, sample discharge reservoir and cathode buffer reservoir, and 750 V for the anode. Figure 2
Is a 20 bp DN separated by using the above four types of electrophoresis solutions.
The electropherogram (electropherogram) of A is shown. If the MC concentration in the running solution is too low, proper separation cannot be performed, but as the MC concentration increases, fragments up to 500 bp can be sieved better.

[Test Example 2] MC for separation of DNA
Effect of Concentration and Separation Volt Number As the sample, a sample was used in which the DNA having the highest peak at 0.5 μg / ml was diluted with DNAase-free water (ICN Biomedicals). 0.3% (w / w), 0.5% (w /
w), 0.7% (w / w) and 1.0% (w / w) MC (aqueous solution having a viscosity of 2% at 20 ° C., manufactured by Sigma Chemical Co., Ltd.) was added to 50 mM boric acid- It was added to a Tris buffer solution (manufactured by Kanto Kagaku Co., Ltd.) at 100 ° C., and stirred at room temperature until it became uniform and transparent to obtain four kinds of solutions. 0.5 μg / ml EtBr (manufactured by Nippon Gene Co., Ltd.) was added to the obtained solution to obtain four kinds of electrophoretic solutions.

The electrophoresis was carried out using a HITACHI LED detector (with SV1100 microchip electrophoresis apparatus with Hitachi Electrox Engineering Co.). The electrophoresis solution prepared by the above method was used as HITACHI.
It was introduced into the microchannel on the chip using the tube of the electrophoresis chip kit. The sample was applied at 300V for 60 seconds. The number of separation bolts is 1 for the sample, the sample discharge reservoir, and the cathode running liquid reservoir.
Set to 30V, anode is 550 only at the lowest
Electrophoresis was performed at V, and the others were performed at 750V. Figure 3
Shows the electropherogram (electropherogram) of DNA separated by changing the MC concentration and the separation voltage. It can be seen that the low-concentration MC running solution is suitable for the separation of large DNA fragments, and the high-concentration MC running solution is suitable for the small DNA fragments. Therefore, when the components of the DNA sample have base pairs with a wide range of sizes, it can be seen that it is preferable to coexist with the low-concentration and high-concentration electrophoretic solutions.

[Example 1] As an electrophoresis sample of 50 bp DNA under isocratic condition and gradient condition, 50 bp DNA (purchased from Life Technologies) was diluted with DNAase-free water (ICN Biomedicals). I used one. HPMC was added to each of 50 mM boric acid-Tris buffer (manufactured by Kanto Kagaku Co., Ltd.) at 100 ° C., and the mixture was stirred at room temperature until it became uniform and transparent, and 0.5 μg /
mlEtBr (manufactured by Nihon Gene Co., Ltd.) was added, and an electrophoretic solution A containing 0.3% (w / w) of HPMC with respect to the whole,
An electrophoretic solution B containing 0.7% (w / w) HPMC was obtained.
When the microchannel is filled with only the electrophoresis running buffer A,
When the microchannel is filled with only the electrophoretic solution B,
Experiments were carried out separately when 1/3 part by weight of the running solution A was filled in the microchannel and then 2/3 parts by weight of the running solution B was placed in the microchannel.

The electrophoresis was performed using a HITACHI LED detector equipped with an SV1100 microchip electrophoresis device (manufactured by Hitachi Electrox Engineering).
The electrophoretic solution prepared by the above method was introduced into the separation channel on the chip using the tube of the HITACHI electrophoresis chip kit. The sample was applied at 300V for 60 seconds. The separation voltage was set to 130 V for the sample, the sample discharge vial and the cathode migration solution vial, and the anode was run at 550 V. Figure 4
1 shows electropherograms (electropherograms) of small DNA separated under isocratic and gradient conditions. From FIG. 4, the separation of large fragments was poor in the 0.7% HPMC running solution, and the separation of small fragments was poor in the 0.3% HPMC running solution. Therefore, in order to separate DNA fragments having a wide range of sizes from small fragments to large fragments, it is necessary to use 1: 2.
It can be seen that it is preferable to use the 0.7% and 0.3% HPMC mixed electrophoretic solution having the concentration gradient of the ratio (w / w).

[Example 2] As an electrophoretic sample of φx174-HindIII enzyme cleavage fragment under isocratic and gradient conditions, φx174-HindIII enzyme (purchased from Takara Shuzo) cleavage fragment was DNAase-free water (ICN Biomedicals). (Manufactured by the company) was used. MC
Was added to 50 mM boric acid-Tris buffer (manufactured by Kanto Kagaku Co., Ltd.) at 100 ° C., and the mixture was stirred at room temperature until it became uniform and transparent, and 0.5 μg /
mlEtBr (manufactured by Nihon Gene Co., Ltd.) was added, and an electrophoretic solution A containing 0.3% (w / w) MC with respect to the whole was prepared.
An electrophoretic solution B containing 0% (w / w) MC was obtained. Electrophoresis solution A
Filling only microchannels and running solution B
Experiments were carried out separately in the case where only the microchannel was filled with only one part and when the microchannel was filled with ½ part by weight of the running solution A, and then the microchannel was filled with ½ part by weight of the running solution B. .

The electrophoresis was carried out using an SV1100 microchip electrophoresis apparatus (manufactured by Hitachi Electrox Engineering) equipped with a HITACHI LED detector.
The electrophoretic solution prepared by the above method was introduced into the microchannel on the chip using the tube of the chip kit for HITACHI electrophoresis. 60 at 300V
It was applied for 2 seconds. Separation voltage is 130 for sample, sample discharge vial and cathode running solution vial.
It was set to V, and the anode was electrophoresed at 550V. Figure 5
Is φx174-Hind under isocratic and gradient conditions
The electropherogram of the III enzyme cleavage fragment is shown. Baseline separation is only 0.3% MC and 1.0% MC
It was found that gradient conditions were necessary to obtain the separation of both small and large fragments, as it was not sufficient by itself. Furthermore, 1.0% MC and 0.3
Coexistence of% MC in the ratio of 1: 2 improved the separation of 271/281 bp and the baseline separation of 1078/1353 bp, but when the addition amount of 1.0% MC was further increased, small fragments were obtained. Separation of sucrose continued to improve while large fragments began to be lost. These results show that in gradient eluates, different matrix ratios need to be adjusted by the sample for optimal results.

Example 3 As an electrophoresis sample of 100 bp DNA under isocratic and three-step gradient conditions, 100 bp DNA (GenS (GenS
ura) purchased from Laboratories) DNAase-free water (I
The product diluted with CN Biomedicals was used. HPMC was added to each of 50 mM boric acid-Tris buffer (manufactured by Kanto Kagaku Co., Ltd.) at 100 ° C., and the solution was stirred at room temperature until it became uniform and transparent.
0.5 μg / ml EtBr (manufactured by Nippon Gene Co., Ltd.) was added to prepare an electrophoresis solution A containing 0.3% (w / w) of HPMC and 0.5% (w / w) of HPMC. An electrophoretic solution B containing and an electrophoretic solution C containing 0.7% (w / w) HPMC were obtained. 1/3 parts by weight of the running solution A was filled with the running solution A alone, the running solution B was only loaded in the microchannel, and the running solution C was only loaded in the microchannel. Then 1 /
Experiments were carried out separately when 3 parts by weight of the running solution B was filled and finally 1/3 parts by weight of the running solution C was filled in the microchannel.

The electrophoresis was carried out using an SV1100 microchip electrophoresis apparatus (manufactured by Hitachi Electrox Engineering Ltd.) equipped with a HITACHI LED detector.
The electrophoretic solution prepared by the above method was introduced into the microchannel on the chip using the tube of the chip kit for HITACHI electrophoresis. 60 at 300V
It was applied for 2 seconds. Separation voltage is 130 for sample, sample discharge vial and cathode running solution vial.
It was set to V, and the anode was electrophoresed at 550V. Figure 6
Is 100 bpD under isocratic and three-step gradient conditions
The electropherogram of NA is shown. From FIG. 6, 0.
Three-step gradients of 3% MC, 0.5% HPMC and 0.7% HPMC gave better baseline separation of all DNA fragments within 3 minutes compared to the isocratic matrix used alone, respectively. Was given.

[Test Example 3] Reproducibility of migration time of 100 bp DNA fragment under three-step gradient conditions For a DNA fragment of 100 bp to 1000 bp, three-step gradient conditions were set in the same manner as in Example 3, and 6 consecutive times were performed. The sample was injected and the migration time was measured. The results are shown in Table 1. Comparative standard deviation of migration time
rivation: RSD) is less than 0.53%, demonstrating good reproducibility of this method.

[0041]

[Table 1]

[0042]

EFFECTS OF THE INVENTION By using the present invention to easily form a stepwise concentration gradient of a water-soluble matrix in a microchannel on a microchip for electrophoresis, various bp sizes can be obtained as compared with the non-gradient method. Complex DNA consisting of
The separation performance of the sample can be improved. Also,
Since the present invention does not require EOF to form a stepwise concentration gradient, the accuracy of analysis can be improved. Furthermore, the present invention is excellent in reproducibility and therefore excellent in the analysis of DNA and restriction enzyme fragments.

[Brief description of drawings]

FIG. 1 is a diagram showing a microchip.
(A) is a front view of a microchip. FIG. 3B is a plan view of the upper substrate 3 that constitutes the microchip.
(C) is a plan view of the lower substrate 2 that constitutes a microchip.

FIG. 2 shows an electropherogram (electropherogram) of 20 bp DNA separated using the above-mentioned four types of electrophoresis solutions.

FIG. 3 shows an electropherogram (electropherogram) of small DNA separated by changing the MC concentration and the separation voltage.

FIG. 4 shows electropherograms (electropherograms) of DNA separated under isocratic and gradient conditions.

FIG. 5 shows φx17 under isocratic and gradient conditions.
An electropherogram of a 4-HindIII enzyme cleavage fragment is shown.

FIG. 6 shows electropherograms of 100 bp DNA under isocratic and 3-step gradient conditions.

[Explanation of symbols]

R1 to R4 reservoir 1 whole microchip 2 Lower substrate 3 Upper board 4 Sample introduction groove 5 separation grooves 6 through holes 7 intersection

Continued front page    (72) Inventor Tsang Rifa             Tokushima Prefecture Tokushima City Shomachi 1-2 chome             Room 16 F term (reference) 2G045 AA35 DA12 DA13 FB05                 2G052 AA28 AB20 AD46 DA05 ED14                       GA22 JA04 JA09

Claims (8)

[Claims] 1. A device for microchip electrophoresis, wherein a water-soluble polymer is filled in a microchannel on a microchip as an electrophoretic solution with a concentration gradient. 2. The device for microchip electrophoresis according to claim 1, wherein the electrophoretic solution further contains a buffer solution. 3. The device for microchip electrophoresis according to claim 1, wherein the water-soluble polymer is a non-crosslinked water-soluble polymer. 4. The device for microchip electrophoresis according to claim 1, wherein the number of non-crosslinked water-soluble polymers is two or more. 5. The device for microchip electrophoresis according to claim 1, wherein the non-crosslinked water-soluble polymer is hydroxymethyl cellulose and / or methyl cellulose. 6. The concentration of hydroxymethyl cellulose is 0.01 to 2.0% (w / w), and the concentration of hydroxymethyl cellulose is 0.2 to 2.0% (w) with respect to the entire running solution.
/ W) and the volume ratio of both is 0.1 to 10: 0.1.
10. The device for microchip electrophoresis according to claim 1, wherein the device is 10. 7. The concentration of methylcellulose in the whole electrophoretic solution is 0.01 to 0.5% (w / w), and the concentration of methylcellulose is 0.6 to 2.0% (w / w), The volume ratio of both is 0.1-5: 0.2-10, The apparatus for microchip electrophoresis in any one of Claims 1-6 characterized by the above-mentioned. 8. A methylcellulose concentration of 0.01 to 2.0% (w / w), a hydroxymethylcellulose concentration of 0.01 to 0.60, and a hydroxymethylcellulose concentration of 0.61 to 0.61 with respect to the entire running solution. 2.0%
(W / w), the volume ratio of the three is 0.1-5: 0.1-1
It is 0: 0.1-10, It is characterized by the above-mentioned.
The device for microchip electrophoresis according to any one of 1.