JP2004298018A - Method for separating and recovering nucleic acid with array for reaction to convert probe into solid phase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently separating and recovering a nucleic acid with high accuracy by using a probe array and to provide the array for a reaction to convert the probe into a solid phase for separating and recovering the nucleic acid. <P>SOLUTION: The array for the reaction to convert the probe into the solid phase is designed to separate and recover the desired nucleic acid. The array for the reaction to convert the probe into the solid phase comprises the probe converted into the solid phase in a support. Furthermore, the array for the reaction to convert the probe into the solid phase characterized as follows is provided. The support is provided with one or a plurality of individually formed reaction parts. Each reaction part of the support has ≥2 openings and the probe hybridizing with the nucleic acid to be separated and recovered for each reaction part is immobilized in the reaction part. Furthermore, the reaction part is provided with an electrode. The method for separating and recovering the nucleic acid comprising using the array for the reaction to convert the probe into the solid phase is provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for separating and recovering nucleic acids using a probe-immobilized reaction array.
[0002]
[Prior art]
As a conventional method for separating and recovering nucleic acids, a method of cutting out a target band after electrophoresis on a gel such as a differential display method, a subtraction method, and the like are performed. Further, in the method described in Patent Document 1, hybridization is performed using a substrate on which a plurality of types of nucleic acids are immobilized, each of the hybridized nucleic acids is separated, and directly separated from the substrate as it is. A method of recovery is disclosed.
[0003]
However, in the method described in Patent Document 1, since a plurality of types of nucleic acids are immobilized on a support, it is necessary to carefully identify the nucleic acid immobilization position when recovering hybridized nucleic acids after hybridization. Yes, stripping each nucleic acid at the tip of the chip or the like is a complicated operation. In addition, when a DNA microarray substrate is used, it is difficult to identify the position where the nucleic acid is immobilized after the hybridization, so that it is difficult to identify the position where the nucleic acid is immobilized, and there is a risk that the nucleic acid other than the target is recovered.
[0004]
Patent Literature 2 discloses a polynucleotide separation method using a microarray. In this method, a hybridized target is dissociated by heating, but even if the solution is locally heated, There is also a possibility that heat that propagates in the solution will dissociate the target that is hybridized to the surrounding probe.
[0005]
[Patent Document 1]
Japanese Patent Application Laid-Open No. 2002-253238
[Patent Document 2]
JP 2000-279169 A
[Patent Document 3]
Japanese Patent Publication No. 11-509423
[Problems to be solved by the invention]
In view of the above situation, an object of the present invention is to efficiently and accurately separate and recover nucleic acids using a probe array.
[0009]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have completed the present invention based on the bold idea of recovering nucleic acids after analysis, although conventional capillary arrays have been used only for detecting nucleic acids. Reached. Specifically, using a probe-immobilized reaction array having a reaction portion such as a capillary shape, a nucleic acid is hybridized to the immobilized probe, and the probe immobilized is capable of dissociating the hybridized nucleic acid. A phase reaction array was developed to achieve the purpose.
[0010]
That is, in one embodiment of the present invention, a probe-immobilized reaction array for separating and recovering a desired nucleic acid,
The probe-immobilized reaction array is a probe-immobilized reaction array in which a probe is immobilized on a support,
The support includes one or a plurality of reaction units independently formed on the support,
Each reaction part of the support has two or more openings,
In the reaction section, a probe that can hybridize with a nucleic acid to be separated and recovered for each reaction section is immobilized,
The reaction unit includes an electrode,
The present invention provides a probe-immobilized reaction array characterized in that:
[0011]
Further, the present invention provides the probe-immobilized reaction array, wherein the reaction section has a capillary shape.
[0012]
Further, the probe-immobilized reaction array,
The two or more openings are a single outlet and a plurality of solution inlets,
The injection port is located at one end of the capillary in the reaction section,
A main flow path is formed from the injection port toward the other end of the capillary,
A plurality of branch channels are formed from the end of the main channel,
At each end of the branch channel, the solution fractionation port is formed,
The present invention provides a probe-immobilized reaction array characterized in that:
[0013]
Further, the probe-immobilized reaction array,
The two or more openings are one inlet, one outlet, and a plurality of solution sampling ports,
The inlet is located at one end of a capillary in the reaction section,
The outlet is located at the other end of the capillary in the reaction section,
A main flow path is formed from the inlet toward the outlet,
In the main flow path, a plurality of branch flow paths are formed, and the branch flow path is formed on the outlet side than any probe fixed to the main flow path,
At each end of the branch channel, the solution fractionation port is formed,
The present invention provides a probe-immobilized reaction array characterized in that:
[0014]
Furthermore, the present invention provides the probe-immobilized reaction array, wherein the reaction section has a well shape.
[0015]
Furthermore, the present invention provides the probe-immobilized reaction array, wherein the electrodes are independently provided at probe-immobilized positions.
[0016]
Furthermore, the present invention provides the probe-immobilized reaction array, wherein the reaction section further includes a heater.
[0017]
Furthermore, the present invention provides the probe-immobilized reaction array, wherein the array further includes a heat transfer layer.
[0018]
Further, there is provided the above-described probe-immobilized reaction array, wherein the probes are indirectly immobilized on a support by beads to which the probes are immobilized.
[0019]
Further, the present invention provides the probe-immobilized reaction array, wherein the reaction section has one probe immobilized for each reaction section.
[0020]
Further, the present invention provides the probe-immobilized reaction array, wherein the reaction section has a plurality of types of probes immobilized thereon.
[0021]
In a second aspect of the present invention, a method for separating and recovering nucleic acids in a probe-immobilized reaction array,
1. Injecting a test sample into the reaction section of the probe-immobilized reaction array,
2. Target nucleic acid contained in the test sample, a step of hybridizing with a probe,
3. Removing the test sample not hybridized to the probe,
4. Detecting the hybridized nucleic acid,
5. A step of dissociating and recovering the hybridized nucleic acid,
A method is provided comprising:
[0022]
Also, in the above method,
In the step 1, the probe-immobilized reaction array is an array in which the same probe is immobilized for each reaction part, the test sample is a test sample different for each reaction part, and The test samples are labeled with different types of labeling substances, and different test samples are injected into each reaction section,
In the step 5, as a result of the detection in the step 4, dissociate and collect only nucleic acids in which a difference in hybridization is detected between the test samples;
A method is provided.
[0023]
Further, the above method,
Further, the present invention provides a method comprising determining the base sequence of the recovered nucleic acid.
[0024]
Further, in the above method, the dissociation of the nucleic acid provides a method of being electrochemically dissociated.
[0025]
Further, in the above method, the dissociation of the nucleic acid provides a method by heating.
[0026]
Further, there is provided the above method, wherein in the step 5, a voltage is independently applied to each probe-immobilized region, and a target nucleic acid dissociated for each probe is collected.
[0027]
BEST MODE FOR CARRYING OUT THE INVENTION
As used herein, the term "nucleic acid" refers to various naturally occurring DNAs and RNAs, as well as artificially synthesized peptide nucleic acids, morpholino nucleic acids, methylphosphonate nucleic acids and S-oligonucleic acids. Refers to nucleic acid analogs and the like.
[0028]
The term "target nucleic acid" as used herein refers to a nucleic acid to be detected by a probe. Generally, a nucleic acid probe is designed to have a base sequence complementary to a target nucleic acid. When the test nucleic acid contained in the test sample has the base sequence of the target sequence, hybridization occurs between the nucleic acid probe and the target sequence. Therefore, by detecting this hybridization, it is possible to analyze the nucleic acid contained in the test sample. Hybridization may be detected by a means known per se. The base sequence that becomes the target of the target nucleic acid is referred to as “target sequence”.
[0029]
As used herein, the term “test sample” refers to a sample prepared from a biological sample such as cells, tissues, organs, blood, serum, lymph, tissues, hair and earwax collected from an individual as desired. And a sample that is to be subjected to a test containing a substance that is artificially synthesized or manufactured. Further, the “test sample” may be a sample obtained by subjecting a biological sample to any necessary pretreatment such as homogenation and extraction, if necessary. Such a pretreatment could be selected by one skilled in the art depending on the biological sample of interest.
[0030]
The term "individual" as used herein refers to any mammal, including humans, dogs, cats, cows, goats, pigs, sheep, and monkeys, as well as non-mammalian organisms such as plants and insects.
[0031]
Hereinafter, an embodiment of the probe-immobilized reaction array of the present invention will be described with reference to an array in which a reaction section is a capillary shape (FIG. 1).
[0032]
FIG. 1A is a plan view of a probe-immobilized reaction array of the present invention as viewed from above. The probe-immobilized reaction array 1 is an example of a probe-immobilized reaction array manufactured using a silicon substrate 8 and a transparent glass substrate 9.
[0033]
Inside the substrate of the probe-immobilized reaction array 1, three capillaries including a plurality of capillaries 2 are formed in parallel on the same plane in parallel. Since the capillaries are formed independently of each other, they do not mix with the fluid contained in other capillaries. Openings are formed at both ends of each capillary. Specifically, the capillary 2 is formed with a sample inlet 5 and a sample outlet 6 respectively. An appropriate probe group 3 is fixed inside the capillary 2 according to the nucleic acid to be captured and recovered by hybridization.
[0034]
By using a probe-immobilized reaction array having a plurality of reaction parts like this probe-immobilized reaction array, it becomes possible to perform a reaction on a different test sample for each independent capillary, and to perform the reaction on the same substrate. Can carry out a plurality of reactions.
[0035]
A nucleic acid that specifically binds to a target nucleic acid to be detected is immobilized on each capillary as a probe. Those skilled in the art can easily select a probe to be used according to an analysis item. Further, such a probe can be easily prepared by those skilled in the art. For example, PCR products or synthetic oligonucleotides can be used.
[0036]
As described above, the number of probes immobilized on a single probe-immobilized reaction array is not limited to one, and a plurality of probes may be immobilized depending on the analysis item and the nucleic acid to be recovered. May be. In this case, a plurality of nucleic acids can be hybridized at one time, and the hybridized nucleic acids can be collected, and efficiency can be improved. For example, in one array, it is possible to perform hybridization and recovery of various nucleic acid sequences derived from one individual, and it is possible to reduce errors in handling a test sample between samples.
[0037]
For example, in FIG. 1, hybridization is performed on a test sample derived from one individual in a capillary 2, hybridization is performed on a test sample derived from another individual in another capillary, and the remaining capillaries are used as controls. Neither analysis may be performed.
[0038]
FIG. 1B is a cross-sectional view of the probe-immobilized reaction array 1 according to the present embodiment cut along a capillary 2.
[0039]
The above-described probe-immobilized reaction array 1 can be manufactured, for example, as follows. A silicon substrate and a glass substrate having the same size are prepared, and are referred to as a substrate 8 and a substrate 9. A groove is formed in the substrate 8 by etching. Further, holes are formed in the substrate 8 in accordance with the pattern of the non-conductive portion 4, and the substrate 8 is filled with a non-conductive material.
[0040]
On the other hand, a desired probe is prepared in advance according to the target nucleic acid. These probes may be fixed by spotting the bottom of the groove of each reaction section for each reaction section. In the substrate 9, through holes are formed in portions corresponding to both ends of the groove of the substrate 8. Next, the substrate 8 and the substrate 9 are joined. Bonding portions 5a and 6b are formed by bonding a glass tube of a desired length to each opening, and a capillary is formed. The size of the space of the capillary shape can be set to various sizes according to the application, but practically, the width is 10 μm to several mm, the depth is 1 μm to 500 μm, the length is several mm to 100 mm, and the capillary interval is About 10 μm to several mm is sufficient. However, considering the efficiency of the reaction, since the diffusion rate of mRNA to be measured or nucleic acid such as cDNA converted from mRNA is as low as several μm per second, the cross-sectional shape of the capillary-shaped space should be wide. By adopting a flat structure with a shallow depth, it can be expected to shorten the reaction time, reduce the amount of sample, increase the observation field of view, and the like.
[0041]
In the above example, the groove is formed in the substrate 8, but the groove may be formed in the substrate 9 or in both the substrate 8 and the substrate 9. Further, as a material of the support, a glass substrate was used as a substrate used as a lid, and a silicon substrate was used as a substrate for forming a groove. However, the present invention is not limited to this. A substrate may be used. Further, the two substrates used may be made of the same material. However, the substrate on which the probe is immobilized must be a conductive substrate such as a silicon substrate. Further, the member to be used may be determined so that the transparent member is arranged in the observation direction. Alternatively, a support formed of a plastic resin, rubber, or the like may be used. Further, a support formed of a material such as glass, silicon, plastic resin and rubber may be used in combination. However, also in this case, the probe-immobilized portion must be a conductive substrate. Further, although a non-conductor is incorporated in the substrate 8 in the drawing, a conductive material may be incorporated in the probe-immobilized portion of the substrate 8 made of a non-conductive material. Further, the non-conductor may not be incorporated in the substrate 8.
[0042]
In the above example, a plate-like substrate is used as the support, but the present invention is not limited to this.
[0043]
In a glass or silicon wafer-like substrate, for example, a groove, a hole for a non-conductor, and a through hole can be formed by a technique such as photolithography-etching. In the case of plastic resin or rubber, grooves, holes for non-conductors and through holes can be formed by machining or molding.
[0044]
As a means for fixing the probe, any means known per se may be used. For example, if the nucleic acid probe is immobilized, a spotting method, an optical solid phase immobilization method, or the like may be used. In the above example, the probe is fixed to the bottom of the groove of the substrate 8, but it may be fixed to the substrate 9 or may be fixed to the side surface of each reaction section. The substrate can be subjected to an appropriate surface treatment for immobilizing the probe, such as a poly-L-lysine treatment, an aminosilane treatment, and an oxide film treatment.
[0045]
Further, as a probe to be immobilized, a probe previously immobilized on beads may be used. In this case, the beads on which the probe is immobilized are immobilized on the substrate. As the beads to be used, for example, beads having an average diameter of 0.05 to 200 μm may be used, and preferably beads having an average diameter of 0.1 to 30 μm. Further, the material of the beads to be used needs to be a conductive material.
[0046]
As means for immobilizing a desired nucleic acid probe on such beads, any means known per se can be used depending on the material of the substrate. For example, it is possible to improve the known means so that they can be applied to curved surfaces, especially spherical surfaces, of solid or granular substrates. For example, in order to dispose a nucleic acid probe on a substrate that has been processed into a curved body, a quantitative method can be applied to a partial region of the curved surface by appropriately combining the two methods of the photosolid phase method and the spotting method. The nucleic acid probe can be solid-phased.
[0047]
In order to immobilize the beads on the substrate, for example, any means known per se may be used. In particular, it is preferable that the beads are magnetic particles, and it is also possible to fix the beads by a magnetic force by previously fixing the magnet at a desired position in the capillary.
[0048]
In addition, after the probe is bound to the bead surface, non-specific binding can be prevented by performing an inactivation treatment. This inactivation treatment is achieved by a method of treating with a partially degraded product of salmon sperm DNA, a random oligonucleotide, bovine serum albumin, or the like, or a method of treating unreacted functional groups with a known method such as ethanolamine.
[0049]
FIG. 1 shows an example in which three spots exist in one capillary. However, the number of spots immobilized on one capillary is not limited to this. Depending on the number of target nucleic acids contained in the test sample, the number of spots may be one or more than one. Therefore, the number of spots may be different for each capillary. It is not necessary that all capillaries included in the probe-immobilized reaction array include a plurality of types of probes, and a control capillary having no immobilized probe may be provided.
[0050]
Next, the joining of the two substrates may be performed before or after fixing the probe by means known per se. For example, when a nucleic acid probe is bonded before immobilization, it is preferable to immobilize the nucleic acid probe by applying a photo-solid phase method (for example, see Japanese Patent Application Laid-Open No. 6-504997). For example, in the case of silicon-quartz glass, an adhesive may be printed by a screen printer and bonded. For example, in the case of silicon-pyrex glass, bonding may be performed at high temperature and high voltage by an anodic bonding method used in a semiconductor process.
[0051]
The probe-immobilized reaction array of the present invention includes electrodes. The electrode is for applying a negative charge to the immobilized probe. Therefore, as described above, the portion to which the probe is immobilized needs to be conductive, and the portion needs to be configured to be able to apply a charge from the electrode. For example, as shown in FIG. 1B, it is preferable that the electrode 7 is fixed on the back side of the probe immobilization site of the conductive substrate. The electrode may cover the array bottom surface, but is preferably an independent electrode for each probe-immobilized site. According to such a structure, the electrodes can be controlled for each probe. However, in this case, it is necessary to incorporate a non-conductive material into the base material so that electricity is not transmitted to portions other than the target probe fixing position (FIG. 1 and the like). With such a structure, it is possible to apply a negative charge only to a region to be collected, and it is possible to separate a nucleic acid hybridized for each probe.
[0052]
Further, it is preferable that the probe-immobilized reaction array of the present invention can control the temperature. The temperature may be controlled by any method. For example, the temperature can be controlled by providing the heater 10 as shown in FIG. Alternatively, the temperature may be externally controlled by providing a heat transfer unit in the array. FIG. 1 shows a case where one heater 10 is used to control the temperature of the entire array. However, an independent heater may be provided for each reaction unit, and temperature control may be performed for each reaction unit.
[0053]
Further, in the present embodiment, an example in which the electrode and the heater are separate from the reaction array according to the present invention has been described. However, the electrode, the heater, the sensor, and necessary wirings and the like may be incorporated in the reaction array. .
[0054]
In addition to the capillary-shaped probe-immobilized reaction array, a micro-liquid reaction device having a sample-shaped inlet and a sample outlet, which has a container-shaped reaction part for enabling a PCR reaction (Patent Document 1) Can be used as the probe-immobilized reaction array of the present invention. For example, the device may have a structure including electrodes.
[0055]
The reaction section provided in the probe-immobilized reaction array according to the present invention refers to a region in the region where a reaction and processing intended by the user such as a chemical reaction or a biochemical reaction are performed. Therefore, in addition to the above shape, for example, the shape of the reaction portion may be a well shape in which the bottom surface is a square, a circle, or an ellipse. Further, the area of the bottom surface and the area of the ceiling surface of the reaction section may be equal or different. The “well shape” used herein does not mean, for example, a form in which the bottom surface and the ceiling surface of the reaction section are spread only in a specific direction as in a capillary shape, but instead forms a bottom surface or a ceiling surface. It refers to a shape having a shape that shows a certain degree of spread in any of the dimensional directions, and has a concave portion or a porous portion that is divided so that a plurality of probe fixing portions can be collectively processed as a group (for example, And Japanese Patent Application Laid-Open No. 9-504864).
[0056]
The probe-immobilized reaction array of the present invention may have various device structures in which reaction parts physically separated from each other are formed. In the example shown in FIG. 1, the reaction unit included in the probe-immobilized reaction array 1 has three reaction units, and one or more openings that can be used as a sample inlet and / or a sample outlet are formed in each of the reaction units. However, the present invention is not limited to this number, and it is sufficient that at least one reaction unit is provided. That is, the reaction units may be independent of each other. For example, a lid or a bottom having an opening may be arranged in a region partitioned by a concave or a convex to form a reaction unit having an effective volume.
[0057]
Further, the shape of the reaction section may have various structures other than the linear structure as shown in FIG. For example, as shown in FIG.
A single outlet and a plurality of solution inlets are formed,
The spout is located at one end of the capillary,
A main flow path is formed from the injection port toward the other end of the capillary,
From the end of the main channel, a plurality of branch channels are formed,
Each end of the branch flow path may have a shape in which a solution sampling port is formed. In the array of FIG. 2, it is preferable that the same cross-sectional shape and the same length are used in order to keep the branch pressure and the flow rate of each branch flow path constant. However, in FIG. 2, the flow path shape is a straight line, but may be a curved line. In FIG. 2, since the branch flow path radiates linearly and extends, the size is small. Also, each sorting port is arranged in an arc on the same circumference. The region from the wavy line to the wavy line in the figure is the reaction region. The electrode is preferably provided independently on the bottom surface of this region for each probe immobilization site.
[0058]
When a capillary array having a shape as shown in FIG. 2 is used, injection and discharge are performed from one location of the injection / discharge port 15, and the fractionation port 18 is used only for fractionation. Therefore, since the flow path is shared by the injection and discharge, the processing capacity is increased, and quick recovery is possible.
[0059]
Further, for example, as shown in FIG.
One inlet, one outlet, and a plurality of solution dispensing ports are formed,
The inlet is located at one end of the capillary,
The outlet is located at the other end of the capillary,
A main channel is formed from the inlet to the outlet,
In the main flow path, a plurality of branch flow paths are formed, and the branch flow path is formed closer to the outlet than any probe fixed to the main flow path,
Each end of the branch flow path may have a shape in which a solution sampling port is formed. Also in the array of FIG. 3, it is preferable that the cross section and the length are the same as in the array of FIG. Further, it is preferable that electrodes are provided as in FIG. Further, in the array of FIG. 3, separate branch flow paths are arranged at different positions of the main flow path, separately from the central drain main flow path. Thereby, the fractionation timing can be performed independently for each branch flow path, or a plurality of times can be performed simultaneously. Also, it can be collected from two or more sampling ports at the same time.
[0060]
When a capillary array having a shape as shown in FIG. 4 is used, the branch flow paths are alternately branched on the branches, so that there is effectively prevented the possibility that contamination is caused by being mixed into another branch flow path by mistake. can do.
[0061]
Further, according to the aspect of the present invention, not only a closed reaction array in which a reaction portion consisting of a lumen is formed inside a substrate, but also a plurality of reaction portions independent of each other and arranged in parallel in an arbitrary direction on a support. If it is provided, even for a container simply partitioned by concave or convex portions, or even in a region without a special partition consisting of a plane, the liquid is sufficiently separated or provided by a large number of vertical holes. Since diffusion is prevented, they can be used in embodiments of the present invention if they are independent of each other. Such a device and its use are also within the scope of the present invention.
[0062]
In addition, the probe-immobilized reaction array of the present invention is preferably in a form that can be used particularly in an automated apparatus. For example, an automation device as shown in Japanese Patent No. 2002-034197 can be used. In such a form, the steps from injection of the test solution and the like to washing and recovery can be performed automatically.
[0063]
By using the probe-immobilized reaction array of the present invention, the nucleic acid can be separated and recovered without complicated operations.
[0064]
Hereinafter, a method for separating and recovering nucleic acids using the probe-immobilized reaction array of the present invention will be described with reference to FIG. As an example, a case where a probe having a known sequence is used to separate and recover a nucleic acid that hybridizes to the probe will be described.
[0065]
FIG. 4 is a schematic diagram of a method for separating and recovering a desired nucleic acid in the probe-immobilized reaction array of the present invention. FIG. 4 shows a case where nucleic acids are separated and recovered using the capillary 2 of FIG.
[0066]
First, a test sample solution containing a target nucleic acid is injected from the injection port 5.
[0067]
The test sample solution to be used needs to contain nucleic acid purified to the extent that a hybridization reaction is possible. For example, tRNA is extracted from a desired tissue or cell, purified directly or as mRNA, and then labeled as described below. In addition, a nucleic acid can be purified from an individual whose hybridization analysis is desired using a commercially available kit or the like.
[0068]
As described above, it is desirable that the test sample containing the target nucleic acid is provided with a traceable optical or physical label so that subsequent detection is possible. Therefore, it is conceivable to use a traceable labeled molecule as described above, for example, a nucleic acid labeled with a fluorescent dye, a luminescent dye, or the like as the test sample. The labeling of the test sample containing the target nucleic acid may be performed, for example, when amplifying the nucleic acid of the test sample injection by PCR using random primers or the like, by adding fluorescently labeled deoxynucleotide triphosphate (dUTP) at the time of nucleic acid synthesis. A method of performing labeling while taking in, or a method of introducing a labeled substance by performing amplification with a primer that has been fluorescently labeled in advance can be used. In addition, a method of binding a dye after amplification can be used, but is not limited thereto. Usable labels include, but are not limited to, Cy3, FITC, and biotin. When a method for detecting a change in polarization after hybridization can be used as a method for detecting hybridization, the target nucleic acid can be used without labeling.
[0069]
As the target nucleic acid, not only one kind of target nucleic acid but also two or more kinds of target nucleic acids can be mixed. In this case, the target nucleic acid may be labeled with several types of labels. Further, the reaction may be performed by changing the type of label for each reaction part.
[0070]
Next, the target nucleic acid contained in the test sample is hybridized to the probe.
[0071]
For example, in the probe-immobilized reaction array 1 in FIG. 1, hybridization is performed under reaction conditions suitable for the probes immobilized in each reaction part. For the hybridization conditions, for example, the temperature may be controlled for each capillary. Further, the reaction solution used at the time of hybridization may be adjusted. All reaction sections may be maintained at a constant temperature to obtain optimum reaction conditions depending on ion concentration and salt concentration.
[0072]
For example, when performing nucleic acid hybridization, even when the optimal concentration in each reaction part is different, by adjusting the salt concentration of a buffer solution or the like used as a solution of a test sample for each reaction part. It is possible to achieve. Further, when the components of the solution used in the actual hybridization reaction contain a DNA denaturant or the like, it is necessary to further adjust the salt concentration and the like as appropriate.
[0073]
Generally, in a hybridization reaction, a reaction between a target nucleic acid and a nucleic acid probe is carried out at a constant temperature of about 45 ° C. to about 65 ° C. for 1 hour to overnight. It is possible to change the conditions. Appropriate reaction conditions to be used will be readily selected by those skilled in the art.
[0074]
Next, the test sample solution that has not been hybridized is removed. For example, a washing operation is performed using a plate washer or the like. By performing such a washing operation, unbound target nucleic acid is removed (upper part in FIG. 4). Thereafter, if necessary, a washing operation is performed with a washing solution having an appropriate salt concentration.
[0075]
Here, when the probe is immobilized via the bead and the bead is a magnetic particle and the bead is immobilized by a magnet, the bead can be suspended and washed. For example, the beads may be suspended by separating the magnet from the array, and after washing, the beads may be immobilized again by the magnet.
[0076]
Next, the nucleic acid hybridized with the probe is detected. For example, for each reaction part, the presence or absence or amount of the labeling substance remaining on the array can be determined by an appropriate measuring means and data processing means while classifying the labeling substance by the position of the labeling substance or the type of the labeling substance. In particular, it is preferred that hybridization can be observed while still contained in the support. For example, by labeling the test sample with a visually recognizable label (such as a fluorescent dye) as described above, the fluorescence of the target substance bound to the probe can be visually recognized as it is. Not only the fluorescence measurement but also the detection may be performed by electrochemical luminescence, color reaction, change measurement, or the like. For example, when the target nucleic acid is used without labeling, a method for detecting a change in polarization after hybridization can be used as a method for detecting hybridization.
[0077]
Next, the hybridized nucleic acid is dissociated from the probe, and the nucleic acid is recovered. Dissociation of the nucleic acid from the probe can be performed by negatively charging the probe.
[0078]
Also, dissociation can be achieved by heating the probe in addition to charging. Heating to a temperature near the Tm of the probe prevents nonspecific hybridization and facilitates dissociation of the nucleic acid by charging. For example, when electrodes are independently provided for each probe-immobilized region as in the probe-immobilized reaction array shown in FIGS. 1 and 4, each probe is charged to dissociate the target nucleic acid. It is possible to collect nucleic acids separately. For example, as shown in FIG. 4, a washing solution or the like may be flowed while sequentially negatively charged from the probe closest to the outlet, and the washing solution or the like containing dissociated nucleic acids may be collected from the outlet. As shown in FIG. 4, by collecting the probe in order from the probe closest to the outlet, other nucleic acids do not pass over the hybridized nucleic acid, so that contamination can be effectively prevented.
[0079]
Next, a method for identifying a gene having a difference in gene expression frequency between test samples using the probe-immobilized reaction array of the present invention will be described. This method enables identification of only genes having a difference in gene expression frequency without previously identifying a large number of probe DNA base sequences.
[0080]
First, a test sample is prepared. For example, when analyzing gene expression frequency for two types of test samples, tRNA is extracted from a desired tissue or cell and from a normal tissue or cell serving as a control, and is directly or purified to mRNA, as described above. The test sample is labeled as described above. Further, the nucleic acid can be purified using a commercially available kit or the like. At this time, labeling is performed with a different labeling substance.
[0081]
Next, each test sample is injected into a separate reaction section on which the same probe is immobilized.
[0082]
Next, hybridization is performed with the probe as described above to remove the unhybridized test sample.
[0083]
Thereafter, the amount of hybridization of both test samples is measured based on the label. If both test samples are labeled with different fluorescent substances, the fluorescence is measured at two kinds of fluorescent wavelengths corresponding to each fluorescent substance.
[0084]
In selecting a probe having a difference in expression level between the two test samples, it is necessary to correct the concentrations of the two types of test samples used. This correction can be performed using, for example, a housekeeping gene considered to be constantly expressed, such as the actin gene. Differences resulting from differences in the concentrations, labeling efficiencies, dyes, etc. of the two test sample DNAs are corrected in advance. After the correction, only those having a difference in the amount of hybridization are selected.
[0085]
Next, as described above, the test sample DNA captured by the hybridization reaction is dissociated from the probe array and collected. The obtained nucleic acid can be recovered by using an ethanol precipitation method or the like. When the test sample DNA has a polyA sequence, it can be recovered using, for example, a column or bead carrier on which a complementary polyT sequence or oligo dT sequence is immobilized.
[0086]
Next, the nucleotide sequence is determined by a method utilizing single molecule technology or after cloning.
[0087]
The obtained target DNA is expected to contain various gene fragments. For this reason, a plurality of base sequence determinations are required. Using the recently developed single-molecule detection technology, it is also possible to perform rapid sequencing without a cloning operation. For details, reference can be made to Japanese Patent No. 2575270 “Nucleic acid base sequence determination method, single molecule detection method, apparatus and method for preparing sample”.
[0088]
In addition, as a method of identifying a base sequence, the recovered test sample DNA may be cloned using a conventional cloning technique, and then the sequence may be identified using a conventional base sequence determination technique. It is preferable to determine the nucleotide sequence of a plurality of DNA fragments and further confirm that there is a difference in expression frequency by using a technique such as reverse transcription PCR.
[0089]
As an advantage of this method, since a probe DNA whose sequence is unknown can be used, it is also possible to identify a gene that has not been identified so far. After recovery, the cloned DNA can be used for subsequent analysis. For example, even when the recovered DNA is a part of a certain gene, a full-length sequence can be obtained by performing colony hybridization using a cDNA library. When the full-length gene has been identified, the gene can be expressed in a host cell such as Escherichia coli or yeast, and the gene can be developed for expression analysis or functional analysis.
[0090]
【The invention's effect】
With the probe-immobilized reaction array of the present invention, a target nucleic acid can be recovered at high speed for a large number of probes, and the amount of a specimen and a sample can be small. High-precision nucleic acid separation and recovery, which has been difficult with conventional DNA microarrays, can be performed. On the other hand, the method of the present invention can detect the difference in the expression level among a plurality of test samples to be compared, separate and collect only the target probe, and determine its nucleotide sequence. Therefore, unlike a conventional hybridization method, a probe having an unknown sequence can be used as a probe to be fixed to an array, and thus, unknown gene analysis can be performed very efficiently.
[0091]
That is, in the conventional nucleic acid probe array, the nucleic acid sequence to be used as a probe needs to be identified. In the present invention, for example, after cloning a cDNA fragment obtained by a shotgun sequence, the nucleotide sequence is determined. Without immobilization, it is possible to perform immobilization directly on the probe array for analysis.
[Brief description of the drawings]
FIG. 1 is a diagram showing a probe immobilization reaction array according to one embodiment of the present invention.
FIG. 2 is a diagram showing a probe immobilization reaction array according to an embodiment of the present invention.
FIG. 3 is a diagram showing a probe immobilization reaction array according to an embodiment of the present invention.
FIG. 4 is a schematic diagram of a method for separating and recovering nucleic acids using the probe-immobilized reaction array of the present invention.
[Explanation of symbols]
1 Probe-immobilized reaction array 2, 12, 22 Capillary (reaction part)
3, 13, 23 Probe group 4, 14, 24 Non-conductor 5, 15, 25 Sample inlet 6, 15, 29 Sample outlet 7 Electrode 8, 9 Substrate 10 Heater 5a, 6b Connecting part 16, 26 Main flow path 17 , 27 Branch flow path 18, 28 Separation port 30 Target nucleic acid

Claims (17)

A probe-immobilized reaction array for separating and recovering a desired nucleic acid,
The probe-immobilized reaction array is a probe-immobilized reaction array in which a probe is immobilized on a support,
The support includes one or a plurality of reaction units independently formed on the support,
Each reaction part of the support has two or more openings,
In the reaction section, a probe that can hybridize with a nucleic acid to be separated and recovered for each reaction section is immobilized,
The reaction unit includes an electrode,
A probe-immobilized reaction array, comprising: The probe-immobilized reaction array according to claim 1, wherein the reaction section has a capillary shape. The probe-immobilized reaction array according to claim 2, wherein
The two or more openings are a single outlet and a plurality of solution inlets,
The injection port is located at one end of the capillary in the reaction section,
A main flow path is formed from the injection port toward the other end of the capillary,
A plurality of branch channels are formed from the end of the main channel,
At each end of the branch channel, the solution fractionation port is formed,
A probe-immobilized reaction array, comprising: The probe-immobilized reaction array according to claim 2, wherein
The two or more openings are one inlet, one outlet, and a plurality of solution sampling ports,
The inlet is located at one end of a capillary in the reaction section,
The outlet is located at the other end of the capillary in the reaction section,
A main flow path is formed from the inlet toward the outlet,
In the main flow path, a plurality of branch flow paths are formed, and the branch flow path is formed on the outlet side than any probe fixed to the main flow path,
At each end of the branch channel, the solution fractionation port is formed,
A probe-immobilized reaction array, comprising: The probe-immobilized reaction array according to claim 1, wherein the reaction section has a well shape. The probe-immobilized reaction array according to any one of claims 1 to 5, wherein the electrodes are independently provided at probe-immobilized positions. . The probe-immobilized reaction array according to any one of claims 1 to 6, wherein the reaction section further includes a heater. The probe-immobilized reaction array according to any one of claims 1 to 6, wherein the array further includes a heat transfer layer. The probe-immobilized reaction array according to any one of claims 1 to 8, wherein the probe is indirectly immobilized on a support by beads on which the probe is immobilized. Reaction array. 3. The probe-immobilized reaction array according to claim 1, wherein one probe is immobilized for each reaction section in the reaction section. 4. The probe immobilization reaction array according to any one of claims 1 to 9, wherein a plurality of types of probes are immobilized on the reaction section. A method for separating and recovering nucleic acids in a probe-immobilized reaction array,
1. Injecting a test sample into the reaction section of the probe-immobilized reaction array,
2. Target nucleic acid contained in the test sample, a step of hybridizing with a probe,
3. Removing the test sample not hybridized to the probe,
4. Detecting the hybridized nucleic acid,
5. A step of dissociating and recovering the hybridized nucleic acid,
A method comprising: The method according to claim 12, wherein
In the step 1, the probe-immobilized reaction array is an array in which the same probe is immobilized for each reaction part, the test sample is a test sample different for each reaction part, and The test samples are labeled with different types of labeling substances, and different test samples are injected into each reaction section,
In the step 5, as a result of the detection in the step 4, dissociate and collect only nucleic acids in which a difference in hybridization is detected between the test samples;
The method characterized by the above. 14. The method according to claim 13, wherein
Further, a method comprising determining the base sequence of the recovered nucleic acid. 13. The method according to claim 12, wherein the dissociation of the nucleic acid is electrochemically dissociated. 13. The method according to claim 12, wherein the dissociation of the nucleic acid is electrochemical dissociation and dissociation by heating. 13. The method according to claim 12, wherein, in the step 5, a voltage is independently applied to each probe-immobilized region to recover a target nucleic acid dissociated for each probe.

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