JP4657524B2 - Microchip electrophoresis apparatus and electrophoresis method using the same - Google Patents

Description

【００４２】
【発明の効果】
本発明を用いて電気泳動用マイクロチップ上のマイクロチャンネル内に水溶性マトリックスの段階的な濃度勾配を簡便に形成させることにより、無勾配な方法と比べて、様々なｂｐサイズからなる複合ＤＮＡサンプルの分離性能を向上させることができる。また、本発明は段階的な濃度勾配の形成にＥＯＦを必要としないことから、分析の精度を向上させることができる。さらに、本発明は再現性が高いことから、ＤＮＡおよび制限酵素断片の分析に優れている。
【図面の簡単な説明】
【図１】図１は、マイクロチップを示す図である。
（Ａ）はマイクロチップの正面図である。
（Ｂ）はマイクロチップを構成する上側の基板３の平面図である。
（Ｃ）はマイクロチップを構成する下側の基板２の平面図である。
【図２】図２は、上記４種類の泳動液を用いて分離した２０ｂｐＤＮＡの電気泳動図（エレクトロフェログラム）を示している。
【図３】図３は、ＭＣ濃度および分離ボルト数を変化させて分離した小ＤＮＡの電気泳動図（エレクトロフェログラム）を示している。
【図４】図４は、無勾配条件下および勾配条件下で分離したＤＮＡの電気泳動図（エレクトロフェログラム）を示している。
【図５】図５は、無勾配および勾配条件下でのφｘ１７４−ＨｉｎｄIII酵素切断断片のエレクトロフェログラムを示す。
【図６】図６は、無勾配および三段階の勾配条件下での１００ｂｐＤＮＡのエレクトロフェログラムを示す。
【符号の説明】
Ｒ１〜Ｒ４ リザーバ
１ マイクロチップの全体
２ 下側の基板
３ 上側の基板
４ 試料導入溝
５ 分離溝
６ 貫通孔
７ 交差部[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for microchip electrophoresis. More specifically, a microfluidic microchip for electrophoresis (hereinafter abbreviated as a microchannel) is filled with an electrophoretic liquid, and a water-soluble polymer is a component of the electrophoretic liquid, and the microchip is water-soluble. The present invention relates to an apparatus for microchip electrophoresis, wherein the conducting polymer has a stepwise concentration gradient.
[0002]
[Prior art]
Capillary electrophoresis (hereinafter abbreviated as CE) is widely used for nucleic acid separation, and it is clear that it is effective for analysis of polymerase chain reaction (PCR) products, separation of restriction enzyme fragments and DNA sequencing. (E.g. Cohen, AS; Najarian, D .; Smith, J, A .; Karger, BLJ Chromatogr. 1988, 458, 323-333, Kheterpal, I .; Mathies, RAAnal. Chem. 1999, 31A-37A Heller, C. Electrophoresis, 2001, 22, 629-643, etc.). For this reason, capillary electrophoresis plays an important role in the Human Genome Project (HGP).
However, in recent years, the development of microelement manufacturing technology and the development of the electronics industry has made it possible to reduce the size of analytical instruments. As a form that can be expected to reduce the consumption of the sample, as shown in DJ Harrison et al./Science 261 (1993) 895-897 and Anal. Chim. Acta283 (1993) 361-366, A chip (particularly a microchip) formed by bonding substrates is proposed.
[0003]
In conventional microchip electrophoresis, DNA samples are generally separated by a single matrix at a fixed concentration. Therefore, when the sample DNA is a complex DNA composed of various bp (base pair) fragments, the separation ability between small fragments and large fragments is not always satisfactory. It was difficult to separate and collect. The same problem is seen in CE, and in order to solve it, for example, a method of mixing several kinds of polymers in a buffer solution (for example, Liang, DH; Chu, B. Electrophoresis, 1998, 19, 2447-2453, Liang , DH; Song, LG; Zhou, SQ; Zaitsev, VS; Chu, B. Macromolecules, 1999, 20, 6326-6332, etc.) and methods using electric field gradients (eg Luckey, JA; Smith, LMAnal. Chem. 1993) 65, 2841-2850, Endo, Y .; Yoshida, C .; Baba, YJ Biochem. Biophys. Methods 1999, 41, 133-141, etc.) have been proposed. However, the stepwise concentration gradient of the polymer solution in CE was possible only in the presence of electroosmotic flow (EOF) (eg, Chen HS .; Chang HT. J. Chromatogr. A, 1999). 853,337-347, Chen HS .; Chang HT.J. Chromatogr. A, 1999, 853, 337-347, etc.).
When the inner surface of the capillary is negatively charged, positively charged ions and particles gather near the inner wall of the capillary, so when an electric field is applied, ions and particles flow toward the negative electrode near the inner surface of the capillary. End up. EOF is the reverse flow that occurs in the center of the capillary to correct the flow.
[0004]
The speed at which the sample is carried by the EOF is about several μm / millisecond, but the sample region (also referred to as a sample plug) moves several μm to several tens of μm due to a delay in switching of the high voltage. In general, the size of the sample plug is several tens of μm, and this causes a problem that undesired phenomena such as the spread of the sample plug and a decrease in analysis accuracy are induced due to the movement of the sample plug due to the delay in switching the high voltage. there were.
[0005]
[Problems to be solved by the invention]
In view of the above-mentioned problems, the present invention can improve the resolution and analysis accuracy of a sample without the presence of EOF by easily forming a stepwise concentration gradient of a water-soluble polymer matrix with a microchip for electrophoresis. Objective.
[0006]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have succeeded in producing a microchip electrophoresis device characterized in that a water-soluble polymer is filled in a microchannel on a microchip with a concentration gradient. As a result of further finding out that the resolution of the sample is improved and the detection accuracy is improved even in the absence of EOF, the present invention has been completed.
[0007]
  That is, the present invention
(1) A microchip electrophoresis apparatus characterized in that a water-soluble polymer is filled in a microchannel on a microchip with a concentration gradient as an electrophoresis solution,
(2) The microchip electrophoresis apparatus according to the above (1), wherein the electrophoresis solution further contains a buffer solution,
(3) The device for microchip electrophoresis according to the above (1) or (2), wherein the water-soluble polymer is a non-crosslinked water-soluble polymer,
(4) It is characterized in that there are two or more kinds of non-crosslinked water-soluble polymers.Above (3)The device for microchip electrophoresis as described,
(5) The non-crosslinked water-soluble polymer is hydroxymethyl cellulose and / or methyl cellulose(3) or (4) aboveThe device for microchip electrophoresis as described,
(6) The concentration of hydroxymethylcellulose relative to the entire electrophoresis solution is 0.01 to 2.0% (w / w), and the concentration of hydroxymethylcellulose is 0.2 to 2.0% (w / w). The volume ratio is 0.1-10: 0.1-10Above (5)The device for microchip electrophoresis as described,
(7) Methyl cellulose for the entire electrophoresis solutionTheConcentration of 0.01-0.5% (w / w), concentration of methylcellulose is 0.6-2.0% (w / w), and the volume ratio of both is 0.1-5: 0 .2-10Above (5)The device for microchip electrophoresis as described,
(8) The concentration of methylcellulose with respect to the entire electrophoresis solution is 0.01 to 2.0% (w / w), the concentration of hydroxymethylcellulose is 0.01 to 0.60, and the concentration of hydroxymethylcellulose is 0.61 to 2. At 0% (w / w), the volume ratio of the three is 0.1-5: 0.1-10: 0.1-10Above (5)The device for microchip electrophoresis as described,
(9) A microchip electrophoresis method using a microchip electrophoresis apparatus in which a water-soluble polymer is filled with a concentration gradient in a microchannel on a microchip as an electrophoresis solution,
(10) The microchip electrophoresis method according to (9) above, wherein electrophoresis solutions having different concentrations of water-soluble polymers are filled in the microchannel one by one in order.
About.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Microchip electrophoresis is particularly effective for separating DNA, for example, by performing electrophoresis in a groove having a width and depth of, for example, about several to 100 μm formed on a microchip widely used in the field of electronics. It is a technique. Since the length of the separation channel used for separation is often several centimeters, the electrophoresis time is usually performed in a short time (several seconds to several tens of seconds). A microchip has a high degree of freedom in circuit design, and since it is easy to make a multicapillary by forming a plurality of flow paths side by side on the same chip, it has an advantage that it is easy to increase the processing capability.
[0009]
As the microchip electrophoresis apparatus in the present invention, a device known per se may be used. Moreover, a commercially available apparatus can also be used, for example, SV1100 microchip electrophoresis apparatus with a HITACHI LED detector (made by Hitachi Electronics Engineering) etc. are mentioned. More specifically, one embodiment of the apparatus will be described below with reference to FIG.
The electrophoresis microchip 1 includes a pair of substrates 2 and 3 made of a glass plate, a quartz plate, or the like (FIG. 1A). On the upper surface of the lower substrate 2, the sample introduction channel 4 and the separation channel 5 are formed so as to intersect with each other (this is referred to as “intersection 7”) (FIG. 1C). A through hole 6 is formed in each of the substrates (hereinafter abbreviated as a cover body) 3 at positions corresponding to the end portions of the grooves 4 and 5 (FIG. 1B). The grooves 4 and 5 are formed on the surface of the substrate 2 by etching.
[0010]
The electrophoretic microchip 1 is formed by the first and second reservoirs R1 and R2 formed by the through holes 6 by bonding the pair of substrates 2 and 3 with the grooves 4 and 5 inside. A sample introduction channel 4 that communicates with each other 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 R <b> 1 to R <b> 4 receives a capillary (capillary tube) for introducing a liquid such as an electrophoretic liquid from the outside, and a voltage is applied to the electrophoretic liquid stored therein. Electrodes (not shown) for applying a voltage are respectively provided.
[0011]
In the microchip for electrophoresis used in the present invention, a microchannel having a width of about 10 to 100 μm and a depth of about 5 to 50 μm is formed by using, for example, laser processing, ion etching processing, or photolithography. For example, it is composed of a pair of substrates made of glass or acrylic. It is considered that the substrate can be selected from those having microchannel formation processing, excellent chemical resistance, and appropriate rigidity. For example, glass, quartz, ceramics, silicon or metal, PDMS It may be a material such as a polymer material such as (polydimethylsiloxane) or PMMA (polymethyl methacrylate). The same applies to the cover body. However, when considering application of optical analysis, a highly transparent material is preferable.
[0012]
The substrate may be a single or multilayer stack. The substrate and the cover body can be laminated and integrated by heat treatment bonding or by means such as adhesion. Further, at the time of lamination, a support body may be laminated and integrated on the side opposite to the cover body in order to reinforce the substrate.
[0013]
In the case where the small hole for sample injection, the small hole for discharge, and the small hole for agent injection are arranged in the cover body, for example, the diameter is several mm or less. Here, it is noted that the agent may be various, such as a solvent for solvent extraction, a reagent for reaction, or a diluent. These small holes can be processed chemically, mechanically, or by various means such as laser processing or ion etching processing.
[0014]
The microchannel can be formed by means such as laser processing or ion etching processing. The length of the microchannel is not particularly limited and can be arbitrarily set as required, 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. Examples of the shape of the microchannel include a cross shape, but other shapes may be used.
[0015]
The microchannel communicates with the outside only through a small hole provided in the cover body such as a small hole for sample injection. For this reason, the fluid circulated in the microchannel is less affected by external conditions and becomes a more stable fine flow. The liquid can be passed through the microchannel through a conduit such as a capillary or a rubber tube. When liquid is passed through a conduit such as a capillary or rubber tube, one end of the conduit is inserted into a small hole for sample injection, a small hole for discharge, or a small hole for agent injection. It becomes possible by attaching and pressing the syringe. In addition, it can be achieved by mechanical means such as a micropump or electric shaking means, or by suction through a small discharge hole.
[0016]
In the present invention, a method of performing electrophoresis by filling an electrophoresis solution containing a water-soluble polymer in the separation channel is preferable. The electrophoresis solution in the present invention contains a water-soluble polymer and a buffer solution, so that a specific higher order is obtained due to the network structure due to the entanglement of the polymer in the electrophoresis solution and the interaction between the water-soluble polymer and DNA. DNA with structure can be isolated. By using this method, separation with different selectivity can be performed only by changing the type or concentration of the water-soluble polymer.
[0017]
As the water-soluble polymer, a non-crosslinked water-soluble polymer is preferably used. Specifically, for example, agarose, hydroxyethyl cellulose, hydroxypropyl methylcellulose (hereinafter abbreviated as HPMC), hydroxycellulose, methylcellulose (hereinafter referred to as MC). Cellulose derivatives such as pullulan and dextran, and polyols such as polyethylene oxide, polyethylene glycol, polypropylene glycol, polyacrylamide, polyvinyl alcohol, polyvinyl pyrrolidone, and polyglycerin. Preferred are hydroxypropyl methylcellulose (HPMC) and methylcellulose (MC).
[0018]
As a buffer solution to be mixed with the water-soluble polymer, for example, a Tris buffer solution, a phthalate buffer solution, a boric acid-Tris buffer solution, or the like can be used, and a commercially available solution may be used. Examples of such commercially available products include boric acid-Tris buffer (manufactured by Kanto Chemical Co., Inc.).
As a method of adjusting the electrophoretic solution by mixing the water-soluble polymer and the buffer solution, a mixture of two or more water-soluble polymers having different concentrations and mixed with the buffer solution, preferably with an equal volume ratio of 1 It is desirable to fill each type. The order of filling the electrophoresis solution may be in order from the highest concentration, or from the lowest concentration. The mixture of the buffer solution and the water-soluble polymer has a higher viscosity as the concentration of the water-soluble polymer is higher, and resistance is generated by the viscosity, so that a stepwise gradient is formed in the microchannel.
The electrophoresis solution having a high concentration of the packed water-soluble polymer and the electrophoresis solution having a lower concentration of the water-soluble polymer that is subsequently packed are in contact with each other within the microchannel, but the diameter of the microchannel is extremely small. Therefore, both of them do not spread and mix. The boundary line between different electrophoresis solutions is visible.
[0019]
The electrophoresis solution containing the water-soluble polymer may contain other components known in the art. Examples of such other components include components that intercalate with DNA. Since this improves the resolution of electrophoresis, it is preferable to contain this component in the present invention. The component that intercalates with DNA usually means a planar molecule that is inserted into a double-stranded DNA such as an acridine dye. In the present invention, it is inserted into a single strand having a higher order structure. It is a concept that includes molecules.
Examples of components that intercalate with DNA include ethidium bromide (hereinafter sometimes abbreviated as EtBr), YO-PRO-1, SYBR Green, and the like. In the present invention, EtBr is preferably used in an amount of about 0.3 to 3.0 μg / ml, preferably about 0.5 to 1.0 μg / ml.
[0020]
The electrophoresis solution is introduced into the microchannel by using a conduit such as the capillary. The water-soluble polymer-containing electrophoresis solution is preferably introduced into the microchannel in order of increasing viscosity. Examples of the conduit include a silica capillary tube and a rubber capillary tube having an inner diameter of about 100 μm, and a capillary included in a commercially available electrophoresis chip kit may be used.
[0021]
Samples that can be used for electrophoresis of the present invention include, for example, nucleic acids including DNA fragments of various sizes, organic molecules such as amino acids, peptides, proteins, metal ions, and the like. Alternatively, a part of the gene amplified by the PCR method and the resulting double-stranded DNA (hereinafter referred to as PCR product) dissociated into single-stranded DNA by heat treatment or the like can be used.
The DNA is preferably diluted with, for example, Dnase-free water or Rnase-free water.
[0022]
The method for injecting the sample into the sample small hole is not particularly limited, and specific examples include an injection method using a syringe, a pressurization method, an electrophoresis method, and the like. The method using a syringe is the same as the method of filling the electrophoresis solution in the microchannel. The pressurization method is a method of injecting a sample by applying 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, a method using a syringe is preferred.
[0023]
In order to observe the electrophoresis migration pattern, a PCR product is labeled with a fluorescent substance and the like, and a method of observing the labeling substance with a marker, and a method of observing the electrophoresis pattern without labeling the PCR product, There is. 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 electrophoresis pattern after electrophoresis. As a staining method, a method known per se may be used, and specific examples include ethidium bromide staining.
Ethidium bromide staining may be performed according to a method known per se (Manitis et al., 1982, Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory).
[0024]
The conditions for electrophoresis may be in accordance with conditions known per se. For example, after injecting a sample into a small hole for injecting a sample, each electrode is inserted, or an electrode formed in advance in each reservoir is used to introduce a sample introduction channel. A high voltage of about 300V is applied to one end of the substrate for a predetermined time, and the other end is grounded (GND) to guide the sample to the intersection. 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 a potential difference, Separated by electrophoresis. Although the electrophoresis time varies depending on the base sequence or temperature of the sample used, it cannot be generally stated, but it is about several seconds to several tens of seconds. In order to prevent diffusion of the sample at the intersection, a weak voltage may be applied to the separation channel when the sample is guided to the intersection, and to the sample introduction channel when the sample is separated.
[0025]
The sample is detected at the detection position. For example, the photomultiplier tube receives light emitted from the contact between the sample components and the luminescent reagent, and an electrical signal corresponding to the intensity of the light is taken out from the photomultiplier tube. The method of doing is mentioned. Alternatively, a UV on-column detector, a diode array detector, a photodiode detector, or a photodiode array detector that measures the absorption spectrum thereof can be used.
[0026]
More specifically, for example, the following procedure is performed using the microchip electrophoresis apparatus. The substrates 2 and 3 of the microchip electrophoresis apparatus are overlapped, and an electrophoresis solution composed of a buffer solution and a water-soluble polymer is introduced from one of the reservoirs (a plurality of reservoirs) from the outside installed in each reservoir. For example, the sample introduction channel 4 and the separation channel 5 are filled with an electrophoretic solution containing a water-soluble polymer by using, for example, a vaccination syringe. Next, a predetermined amount of a small amount of sample is injected into the first reservoir (sample injection point) R1, and an electrophoretic solution (for example, 0.1M Tris-Hou) is injected into the third and fourth reservoirs (agent injection points) R3 and R4. Inject acid buffer). The sample and the electrophoresis solution are injected in the same manner as the method of filling the electrophoresis solution.
[0027]
After injecting the sample, the electrophoresis solution and the water-soluble polymer as described above, electrodes are inserted into the respective reservoirs, or the electrodes formed in the respective reservoirs are used in advance at one end of the sample introduction channel 4 for a predetermined time. Only a predetermined high voltage is applied (for example, 300 V for 60 seconds), and the other end is set to the GND 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 in the traveling direction is the other end. End). As a result, the sample moves through the sample introduction channel 4 and is guided to the intersection 7 after a predetermined voltage application time has elapsed.
[0028]
Next, a voltage of about 550 to 750 V is applied to one end of the separation channel 5 and the other end is switched to the GND potential, and the sample is separated due to a difference in charge, size, and the like. At this time, for example, a voltage of about 130 V is applied to both ends of the sample introduction channel 4 to suppress the diffusion of the sample into the separation channel.
[0029]
After the sample is separated in the separation channel 5 by the above procedure, it is detected by the detector at the detection position on the other end side of the separation channel 5. For example, when migrating in the separation channel 5, the components contained in the sample are separated and reach the fourth reservoir R4 with a time lag. It comes into contact with the luminescent sample input from the third reservoir, causes a chemical reaction in the separation channel 5 and emits light. There is a method in which the emitted light is received by a photomultiplier tube and an electric signal corresponding to the light emission intensity is taken out from the photomultiplier tube. In addition, it is possible to use a photodiode detector that observes a change in absorbance at a wavelength of about 200 to 300 nm or a photodiode array detector that measures the absorption spectrum by utilizing the property that the sample absorbs ultraviolet rays.
In addition, when the DNA is fluorescently labeled, fluorescence emission detection can be performed. Thereby, highly sensitive detection becomes possible. The fluorescence emission detection is preferably performed at an excitation wavelength of about 240 to 600 nm, although it varies depending on the fluorescent substance used. To record the electrophoresis peak, the detector is connected to an integrator.
[0030]
【Example】
[Test Example 1] Effect of MC concentration on separation of 20 bp DNA
As the sample, 20 μg DNA (purchased from GenSura Laboratories) 2 μg / ml diluted with DNAase-free water (ICN Biomedicals) was used. The concentration of MC ((w / w), 0.3% (w / w), 0.5% (w / w), and 0.7% (w / w) of MC An aqueous solution having a viscosity of 2% at 20 ° C., manufactured by Sigma Chemical Co., Ltd., is added to 50 mM boric acid-Tris buffer solution (manufactured by Kanto Chemical Co., Ltd.) at 100 ° C. until the solution becomes uniform and transparent at room temperature. Four types of solutions were obtained by stirring. To each of the obtained solutions, 0.5 μg / ml EtBr (manufactured by Nippon Gene Co., Ltd.) was added to prepare four types of electrophoresis solutions.
[0031]
Electrophoresis was performed using an SV1100 microchip electrophoresis apparatus (manufactured by Hitachi Electronics Engineering) with a HITACHI LED detector. The four types of electrophoresis solutions prepared by the above method were introduced into the microchannel on the chip using the tube of the chip kit for HITACHI electrophoresis.
The sample was applied at 300V for 60 seconds. The number of separation bolts was set to 130 V for the sample, the sample discharge reservoir and the cathode buffer reservoir, and 750 V for the anode.
FIG. 2 shows an electropherogram (electropherogram) of 20 bp DNA separated using the above four types of electrophoresis solutions.
If the MC concentration in the electrophoresis solution is too low, proper separation cannot be performed, but as the MC concentration increases, fragments up to 500 bp can be screened better.
[0032]
[Test Example 2] Effect of MC concentration and number of separated bolts on DNA separation
The sample used was a DNA diluted with DNAase-free water (ICN Biomedicals) at a concentration showing the highest peak at 0.5 μg / ml. MC so that the concentration with respect to the entire electrophoresis solution is 0.3% (w / w), 0.5% (w / w), 0.7% (w / w), and 1.0% (w / w) (An aqueous solution having a viscosity of 2% at 20 ° C., manufactured by Sigma Chemical Co., Ltd.) is added to 50 mM boric acid-Tris buffer solution (manufactured by Kanto Chemical Co., Ltd.) at 100 ° C. until the solution becomes uniform and transparent at room temperature. Four types of solutions were obtained by stirring. 0.5 μg / ml EtBr (manufactured by Nippon Gene Co., Ltd.) was added to the resulting solution to obtain four types of electrophoresis solutions.
[0033]
The electrophoresis was performed using a HITACHI LED detector (SV1100 microchip electrophoresis apparatus manufactured by Hitachi Electronics Engineering Co., Ltd.). The electrophoresis solution prepared by the above method was introduced into the microchannel on the chip using the tube of the chip kit for HITACHI electrophoresis.
The sample was applied at 300V for 60 seconds. The number of separation bolts was set to 130 V for the sample, the sample discharge reservoir, and the cathode electrophoresis reservoir, and the anode migrated at 550 V only when it was the lowest, and 750 V for the other.
FIG. 3 shows an electrophorogram (electropherogram) of DNA separated by changing the MC concentration and the number of separated bolts.
It can be seen that the low concentration MC electrophoresis solution is suitable for separating large DNA fragments, and the high concentration MC electrophoresis solution is suitable for small DNA fragments. Therefore, it can be seen that when the constituents of the DNA sample have base pairs of a wide range of sizes, it is preferable to use a low concentration and a high concentration electrophoresis solution together.
[0034]
[Example 1] Electrophoresis of 50 bp DNA under non-gradient and gradient conditions
As a sample, 50 bp DNA (purchased from Life Technologies) diluted with DNAase-free water (ICN Biomedicals) was used. HPMC was added to 50 mM boric acid-Tris buffer (manufactured by Kanto Chemical Co., Inc.) at 100 ° C., and the solution was stirred at room temperature until it became uniform and transparent, and 0.5 μg / ml EtBr was added to the resulting electrophoresis solution. (Manufactured by Nihon Gene Co., Ltd.), electrophoresis solution A containing 0.3% (w / w) HPMC and electrophoresis solution B containing 0.7% (w / w) HPMC Obtained.
When only the electrophoresis solution A is filled into the microchannel, when only the electrophoresis solution B is filled into the microchannel, 1/3 parts by weight of the electrophoresis solution A is filled into the microchannel, and then 2/3 parts by weight. The experiment was performed separately when the electrophoresis solution B was packed in a microchannel.
[0035]
Electrophoresis was performed using a HITACHI LED detector with an SV1100 microchip electrophoresis apparatus (manufactured by Hitachi Electronics Engineering). The electrophoresis solution prepared by the above method was introduced into the separation channel on the chip using the tube of the chip kit for HITACHI electrophoresis.
The sample was applied at 300V for 60 seconds. The number of separation bolts was set to 130 V for the sample, the sample discharge vial and the cathode electrophoresis solution vial, and the anode was run at 550 V.
FIG. 4 shows electropherograms (electropherograms) of small DNA separated under non-gradient and gradient conditions.
From FIG. 4, the separation of large fragments was poor in the 0.7% HPMC electrophoresis solution, and the separation of small fragments was poor in the 0.3% HPMC electrophoresis solution. Thus, to separate a wide range of DNA fragments from small to large fragments, 0.7% and 0.3% HPMC mixed electrophoresis with a 1: 2 ratio (w / w) concentration gradient can be used. It turns out that it is good to use.
[0036]
Example 2 Electrophoresis of φx174-HindIII Enzyme Cleaved Fragment under No-Gradient and Gradient Conditions
A sample obtained by diluting a cleaved fragment of φx174-HindIII enzyme (purchased from Takara Shuzo) with DNAase-free water (ICN Biomedicals) was used. MC was added to 50 mM boric acid-Tris buffer solution (manufactured by Kanto Chemical Co., Inc.) at 100 ° C., and the solution was stirred at room temperature until it became uniform and transparent, and 0.5 μg / ml EtBr was added to the resulting electrophoresis solution. (Manufactured by Nihon Gene Co., Ltd.), electrophoresis solution A containing 0.3% (w / w) MC and electrophoresis solution B containing 1.0% (w / w) MC with respect to the whole Obtained.
When only the electrophoresis solution A is filled into the microchannel, when only the electrophoresis solution B is filled into the microchannel, 1/2 weight part of the electrophoresis solution A is filled into the microchannel, and then 1/2 weight part of The experiment was performed separately when the electrophoresis solution B was packed in a microchannel.
[0037]
  Electrophoresis was performed using an SV1100 microchip electrophoresis apparatus (manufactured by Hitachi Electronics Engineering) with a HITACHI LED detector. The electrophoresis solution prepared by the above method was introduced into the microchannel on the chip using the tube of the chip kit for HITACHI electrophoresis.
  The sample was applied at 300V for 60 seconds. The number of separation bolts was set to 130 V for the sample, the sample discharge vial and the cathode electrophoresis solution vial, and the anode was run at 550 V.
  FIG. 5 shows an electropherogram of the φx174-HindIII enzyme cleavage fragment under non-gradient and gradient conditions.
  Since baseline separation was not sufficient with only 0.3% MC and 1.0% MC, it was found that gradient conditions were necessary to obtain separation of both small and large fragments. It was.
  Furthermore, coexistence of 1.0% MC and 0.3% MC in a ratio of 1: 2 improved the separation of 271/281 bp and also obtained a baseline separation of 1078/1353 bp. Increasing the MC loading further improved the separation of small fragments while large fragments began to be lost.
  From these results,Electrophoresis solution under gradient conditionsIt can be seen that the ratio of the different matrices needs to be adjusted by the sample to obtain optimal results.
[0038]
[Example 3] Electrophoresis of 100 bp DNA under non-gradient and three-step gradient conditions
As a sample, 100 bp DNA (purchased from GenSura Laboratories) diluted with DNAase-free water (ICN Biomedicals) was used. Each HPMC was added to 50 mM boric acid-Tris buffer (manufactured by Kanto Chemical Co., Ltd.) at 100 ° C. and stirred until the solution became uniform and transparent at room temperature, and 0.5 μg / ml EtBr was added to the resulting electrophoresis solution. (Manufactured by Nippon Gene Co., Ltd.), electrophoresis solution A containing 0.3% (w / w) HPMC, electrophoresis solution B containing 0.5% (w / w) HPMC, and Electrophoresis solution C containing 0.7% (w / w) HPMC was obtained.
When only the electrophoresis solution A is filled in the microchannel, when only the electrophoresis solution B is filled in the microchannel, when only the electrophoresis solution C is filled in the microchannel, and 1/3 parts by weight of the electrophoresis solution A is filled Then, 1/3 parts by weight of the electrophoresis solution B was filled, and finally, 1/3 parts by weight of the electrophoresis solution C was filled in the microchannel.
[0039]
Electrophoresis was performed using an SV1100 microchip electrophoresis apparatus (manufactured by Hitachi Electronics Engineering) with a HITACHI LED detector. The electrophoresis solution prepared by the above method was introduced into the microchannel on the chip using the tube of the chip kit for HITACHI electrophoresis.
The sample was applied at 300V for 60 seconds. The number of separation bolts was set to 130 V for the sample, the sample discharge vial and the cathode electrophoresis solution vial, and the anode was run at 550 V.
FIG. 6 shows an electropherogram of 100 bp DNA under non-gradient and three-step gradient conditions.
From FIG. 6, it can be seen that the three-step gradient of 0.3% MC, 0.5% HPMC and 0.7% HPMC each showed the base of all DNA fragments within 3 minutes compared to the non-gradient matrix used alone. Line separation was better obtained.
[0040]
[Test Example 3] Reproducibility of migration time of 100 bp DNA fragment under three-step gradient conditions
For DNA fragments of 100 bp to 1000 bp, three steps of gradient conditions were set in the same manner as in Example 3, and the sample was injected six times in succession to measure the migration time. The results are shown in Table 1. The relative standard derivation (RSD) of the running time is less than 0.53%, demonstrating that this method has good reproducibility.
[0041]
[Table 1]

[0042]
【The invention's effect】
By using the present invention, a stepwise concentration gradient of a water-soluble matrix is easily formed in a microchannel on a microchip for electrophoresis, so that a composite DNA sample having various bp sizes can be obtained as compared with a non-gradient method. The separation performance can be improved. Further, since the present invention does not require EOF for forming a stepwise concentration gradient, the accuracy of analysis can be improved. Furthermore, since the present invention has high reproducibility, it is excellent for analysis of DNA and restriction enzyme fragments.
[Brief description of the drawings]
FIG. 1 is a diagram showing a microchip.
(A) is a front view of a microchip.
FIG. 4B is a plan view of the upper substrate 3 constituting the microchip.
(C) is a top view of the lower substrate 2 constituting the microchip.
FIG. 2 shows an electropherogram (electropherogram) of 20 bp DNA separated using the above four types of electrophoresis solutions.
FIG. 3 shows an electropherogram (electropherogram) of small DNA separated by changing the MC concentration and the number of separation bolts.
FIG. 4 shows electropherograms (electropherograms) of DNA separated under non-gradient and gradient conditions.
FIG. 5 shows an electropherogram of a φx174-HindIII enzyme cleavage fragment under no gradient and gradient conditions.
FIG. 6 shows an electropherogram of 100 bp DNA under non-gradient and three-step gradient conditions.
[Explanation of symbols]
R1-R4 reservoir
1 Whole microchip
2 Lower board
3 Upper board
4 Sample introduction groove
5 Separation groove
6 Through hole
7 intersection

Claims (10)

  An apparatus for microchip electrophoresis, wherein a water-soluble polymer is filled in a microchannel on a microchip with a concentration gradient as an electrophoretic solution.   2. The microchip electrophoresis apparatus according to claim 1, wherein the electrophoresis solution further contains a buffer solution.   3. The microchip electrophoresis apparatus according to claim 1, wherein the water-soluble polymer is a non-crosslinked water-soluble polymer. The device for microchip electrophoresis according to claim 3, wherein the number of non-crosslinked water-soluble polymers is two or more. The microchip electrophoresis apparatus according to claim 3 or 4 , wherein the non-crosslinked water-soluble polymer is hydroxymethyl cellulose and / or methyl cellulose. The concentration of hydroxymethylcellulose with respect to the entire electrophoresis solution is 0.01 to 2.0% (w / w), the concentration of hydroxymethylcellulose is 0.2 to 2.0% (w / w), and the volume ratio of both is The microchip electrophoresis apparatus according to claim 5, which is 0.1 to 10: 0.1 to 10. The concentration of methylcellulose with respect to the entire electrophoretic solution is 0.01 to 0.5% (w / w), the concentration of methylcellulose is 0.6 to 2.0% (w / w), and the volume ratio of both is 0.1. 6. The microchip electrophoresis apparatus according to claim 5, wherein the ratio is 1 to 5: 0.2 to 10. The concentration of methylcellulose with respect to the entire electrophoresis solution is 0.01 to 2.0% (w / w), the concentration of hydroxymethylcellulose is 0.01 to 0.60, and the concentration of hydroxymethylcellulose is 0.61 to 2.0% ( 6. The microchip electrophoresis apparatus according to claim 5, wherein the volume ratio of the three components is 0.1 to 5: 0.1 to 10: 0.1 to 10 in w / w).   A microchip electrophoresis method using a microchip electrophoresis apparatus in which a water-soluble polymer is filled with a concentration gradient in a microchannel on a microchip as an electrophoresis solution.   The microchip electrophoresis method according to claim 9, wherein electrophoresis solutions having different concentrations of the water-soluble polymer are filled in the microchannel one by one in order.

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