JP4453334B2 - Hybridization apparatus and method - Google Patents

Description

  The present invention relates to a hybridization apparatus and method for hybridizing a single-stranded nucleotide chain and its complementary nucleotide chain.

  Currently, DNA analysis by hybridization using a DNA chip or the like is performed in gene mutation analysis, SNPs (single nucleotide polymorphism) analysis, gene expression frequency analysis, and the like. DNA analysis by hybridization is obtained from a specimen by hybridization with a DNA (DNA probe) of a known base sequence such as a DNA oligo chain or cDNA (Complementary DNA) placed on a substrate such as glass or silicon. The base sequence of DNA (target DNA) is analyzed. In particular, when a DNA chip is used, exhaustive DNA analysis can be performed because a large number of probe DNAs are arranged on one substrate.

  An example of the analysis method using a DNA chip will be briefly described. For example, reverse transcription PCR (Polymerase Chain Reaction) reaction of mRAN extracted from cells, tissues, etc. to DNA probes immobilized on a glass substrate or silicon substrate. In this method, PCR amplification is carried out while incorporating a fluorescent probe dNTP, hybridization is performed on the substrate, and fluorescence is measured with a predetermined detector.

JP-A-11-281534

  By the way, in a normal state, single-stranded DNA in a solution causes hybridization between bases of its own complementary sequence, and has a random coil shape. For this reason, even when trying to hybridize with other single-stranded DNA, steric hindrance is great and it does not bind at high speed. Furthermore, in the case where hybridization has already occurred between the self bases, the complementary sequences may not be 100% bonded to each other and may end incompletely.

  An object of the present invention is to provide a hybridization apparatus and method capable of performing high-speed and reliable hybridization of single-stranded DNA causing steric hindrance.

The hybridization apparatus according to the present invention has a space for storing a solution in which a target nucleotide chain is suspended, and one end of a plurality of probes, which are detection nucleotide chains, is fixed to a surface forming the space. And a plate-like member provided so as to be in contact with the reaction vessel, and formed on the outer surface of the reaction vessel and on the same side as the surface to which one end of the probe is fixed An anode and a plate-like member provided so as to be in contact with the reaction vessel, are formed on the outer surface of the reaction vessel facing the surface to which one end of the probe is fixed, and has a larger surface area than the anode. An AC power source for applying an AC voltage between the cathode, the anode and the cathode, and a surface on the outer surface of the reaction vessel other than the surface on which the anode and the cathode are provided; The temperature adjustment means for adjusting the temperature of the solution stored in the reaction vessel, and until the temperature reaches a temperature higher than the deactivation temperature of the probe having the highest deactivation temperature among the probes that are the nucleotide chains for detection. solution in the reaction vessel was heated, while maintaining the temperature remains the solution temperature was the arrival, by applying a voltage between the anode and the cathode, between the anode and the cathode Binding of a probe and a target that generate self-hybridization by applying an electric field to the solution by generating an electric field of about 1 × 10 ⁶ V / m, 1 MHz, and maintaining the generated electric field for a predetermined time. After the self-hybridization separation process and the separation of the self-hybridization, while cooling the solution in the reaction vessel, And a state of applying a voltage between the anode and the cathode, by alternately repeating the state where no voltage is applied between the anode and the cathode, the target and which is a nucleotide chain as a probe and the target When the temperature of the solution in the reaction container reaches the annealing temperature of an arbitrary probe after completion of the first reaction promotion treatment for promoting extension and hybridization and the first reaction promotion treatment, any and conditions for applying a voltage between the anode and the cathode, by continuing for a predetermined time the state of the annealing temperature while repeating a state in which no voltage is applied alternately between the anode and the cathode A process for promoting hybridization between the probe and the target for all probes contained in the reaction container; After performing the reaction promotion treatment and the second reaction promotion treatment, after cooling the temperature of the solution stored in the reaction vessel to a temperature that does not contribute to a biochemical reaction lower than the annealing temperature of all probes or room temperature in occurs and a state of applying a voltage between the anode and the cathode, and a state where no voltage is applied between the anode and the cathode, the negative pulse electric field between the anode and the cathode By alternately repeating the state in which the voltage is applied, the first target separation is performed by pulling the unreacted target away from the probe and moving it to the surface opposite to the surface on which one end of the probe in the reaction vessel is fixed. After the process and the first target separation process, a voltage is not applied between the anode and the cathode, and an electric field that generates a negative pulse electric field between the anode and the cathode. By alternately repeating the state in which pressure is applied, the unreacted target is pulled away from the probe and moved to a surface opposite to the surface on which one end of the probe in the reaction vessel is fixed. And a controller for controlling the AC power supply and the temperature adjusting means so as to execute a second target separation process for completely removing the target.

In the hybridization method according to the present invention, a solution in which a target nucleotide chain is suspended until reaching a temperature higher than that of the probe having the highest deactivation temperature among the probes that are nucleotide chains for detection. The solution in the reaction vessel having a space portion to be stored and one end of each probe, which is a nucleotide chain for detection, fixed to the surface forming the space portion is heated, and the temperature of the solution is maintained at the reached temperature. An anode formed on the outer surface of the reaction vessel and on the same side as the surface to which one end of the probe is fixed, comprising a plate-like member provided in contact with the reaction vessel And a plate-like member provided in contact with the reaction vessel, and formed on the outer surface of the reaction vessel facing the surface to which one end of the probe is fixed. , By applying a voltage between a cathode having a surface area greater than the ^{anode, 1 × 10 6 V / m} , to generate an electric field of about 1MHz to between the anode and the cathode, was generated A self-hybridization separation step in which an electric field is applied to the solution by maintaining the electric field for a predetermined time to separate the binding of the probe and the target that have caused self-hybridization, and after the separation of the self-hybridization, while the solution in the reaction vessel was cooled, and a state of applying a voltage between the anode and the cathode, by alternately repeating the state where no voltage is applied between the anode and the cathode, the probe A first reaction promoting step for promoting extension and hybridization between the target and the target nucleotide chain, and After completion of the first reaction promoting step, the temperature of the solution in the reaction vessel, when it reaches to the annealing temperature of any of the probe, the conditions for applying a voltage between the anode and the cathode, A process for promoting hybridization between an arbitrary probe and a target by continuing an annealing temperature state for a predetermined time while alternately repeating a state in which no voltage is applied between the anode and the cathode. After the execution of the second reaction promotion step executed with respect to all the probes contained therein and the second reaction promotion step, the temperature of the solution stored in the reaction vessel is lower than the annealing temperature of all the probes. after cooling to a temperature or room does not contribute to the biochemical reaction, and a state of applying a voltage between the anode and the cathode, the anode and the anion Alternately repeating a state in which no voltage is applied between and a state in which a voltage generating a negative pulse electric field is applied between the anode and the cathode, thereby pulling the unreacted target away from the probe, A first target separation step for moving the probe in the reaction vessel to a surface opposite to the surface on which one end of the probe is fixed, and a voltage between the anode and the cathode after the execution of the first target separation step. By alternately repeating a state in which no voltage is applied and a state in which a voltage generating a negative pulse electric field is applied between the anode and the cathode to separate the unreacted target from the probe. And a second target separation step of completely removing the unreacted target by moving it to a surface opposite to the surface to which one end of the substrate is fixed. .

  In the hybridization apparatus and method according to the present invention, the temperature of the solution is raised to a temperature higher than the melting temperature of all the probes, an alternating electric field is applied to the solution, and then the annealing temperature of all the probes is reached or lower. Lower the temperature of the solution.

  In such a hybridization apparatus and method according to the present invention, since the AC electric field is applied while the temperature is higher than the melting temperature, the binding of all the target nucleotide chains (targets) and all the probes is released and the AC electric field is applied. It expands in the direction of the electric field line, and in the expanded state it becomes below the annealing temperature.

  Therefore, in the hybridization apparatus and method according to the present invention, hybridization can be caused at high speed and reliably.

  Hereinafter, a hybridization apparatus to which the present invention is applied will be described.

  A configuration of a hybridization apparatus 10 to which the present invention is applied is shown in FIG. The hybridization apparatus 10 hybridizes a single-stranded nucleotide chain for detection (hereinafter referred to as a probe) with a known base sequence and a target nucleotide chain (hereinafter referred to as a target) detected from a specimen. Device. Hybridization is performed between nucleotide strands of complementary base sequences. For this reason, it is possible to analyze the base sequence of the target by detecting with which probe the target has reacted after this hybridization treatment.

  As shown in FIG. 1, the hybridization apparatus 10 includes a reaction container 12 that forms an internal space in which a buffer solution 11 serving as a hybridization reaction place is reserved. A buffer solution 11 is injected into the reaction vessel 12.

One end of the probe is connected to a predetermined position on the inner side wall of the reaction vessel 12. For example, the end of the probe is modified with a functional group (for example, biotin), and a part of the inner wall of the reactor 12 is also surface-modified with a paired functional group (for example, streptavidin). Then, by bonding the functional groups, one end of the probe can be bonded to a predetermined position in the reaction vessel 12. The number of probes bound in the reaction vessel 12 may be one or plural. Here, it is assumed that two probes P ₁ and P ₂ having different base sequences are connected. In addition, the probe coupling position is not limited to the side wall as long as the position is immersed in the solution 11, and may be the bottom surface.

  For example, when a DNA chip having a plurality of wells is used, only one type of probe is bound in one well (reaction vessel). The present invention can be applied even when such a DNA chip is used.

  The hybridization apparatus 10 also includes a pair of electrodes 13 (for example, a positive electrode 13-1 and a negative electrode 13-2) and a power source 14 that applies power to the pair of electrodes 13. As shown in FIG. 2, the pair of electrodes (the positive electrode 13-1 and the negative electrode 13-2) can apply an electric field to the solution 11 in the reaction vessel 12 when a voltage is applied by the power source 14. The whole is formed in a thin plate shape, for example, and is provided so as to sandwich the reaction vessel 12 from the outside. Furthermore, electric lines of force are generated along the direction from the opposite side of the probe coupling position toward the probe, and further, the distance between the electric lines of force decreases as the probe is approached (the density of electric lines of force is reduced). The size and arrangement of the pair of electrodes 13 are configured so as to be higher. For example, if the probe is coupled to the side wall of the reaction vessel 12, the plus electrode 13-1 is provided on the outer side wall corresponding to the side wall portion to which the probe of the reaction vessel 12 is coupled, and the minus electrode 13-2 is disposed on the opposite side. If the size of the electrode (plus electrode) provided on the outer side wall of the probe and the side wall on the probe side is sufficiently smaller than the size of the opposite electrode (minus electrode 13-2), the above Electric field lines can be formed. The reason why the lines of electric force are formed in this way is that the probe and the target are extended in the solution 11 and the target is electrophoresed to approach the probe.

  Further, the hybridization apparatus 10 includes a temperature sensor 15 that detects the temperature of the solution 11 and a heater / cooler 16 that holds, raises, and lowers the temperature of the solution 11. Since the hybridization apparatus 10 includes the temperature sensor 15 and the heater / cooler 16, the temperature of the solution 11 in the reaction container 12 can be adjusted to a specific temperature.

  The hybridization apparatus 10 includes a controller 17. The controller 17 gives a control command to the heater / cooler 16 to control the temperature of the solution 11 in the reaction vessel 12 and controls the output voltage and current of the power supply 14 to be applied to the solution 11 in the reaction vessel 12. To control the electric field.

  Next, the hybridization procedure by the hybridization apparatus 10 having the above configuration will be described.

  FIG. 3 shows the temperature of the solution 11 in the reaction container 12 at each elapsed time (periods s1 to s9) during hybridization.

  Before starting the hybridization process, as shown in FIG. 4, first, a target (target nucleotide chain) T is dropped in advance in the buffer solution 11. At this time, the temperature in the buffer solution 11 is low, and is a temperature that does not contribute to the biochemical reaction, neither room temperature nor Tm below Tm nor Ta. Further, the electric field in the buffer solution 11 is not applied. Therefore, the probe P and the target T in the solution 11 of the reaction vessel 12 are in a coil shape (supercoil, random coil) in which chains are disturbed in various directions. The probe P is fixed at a predetermined position, but the target T is suspended in the solution 11.

  Subsequently, the heater / cooler 16 is controlled to increase the temperature of the solution 11 in the reaction vessel 12 to a temperature higher than the highest detent temperature Tm-max among the detent temperatures (melting temperatures) Tm of the plurality of probes P. (Period s2).

Here, when heat is applied to double-stranded DNA or RNA stabilized by hydrogen bonding, it is separated into single strands. If all double-stranded state is 100% helix, and all is single-stranded by applying heat is 0% helix, then exactly half of the DNA or RNA is forming a double strand. 50% helix. The temperature at this time is defined as the deny temperature (melting temperature) Tm. The denature temperature Tm is estimated to be high (80 to 90 degrees) if the duplex is stable and low (39 to 40 degrees) if unstable. In general, the melting temperature Tm varies depending on items such as GC base pair content, nucleotide chain length, base pair mismatch (base pairs do not match), salt concentration and composition of the solution 11. Although the melting temperature Tm varies due to these influences, it is empirically estimated that the temperature can be estimated by the following equation.
Tm = 81.5 + (16.6 × logM) + {0.41 × (G + C)}-(0.72 × F)-(500 / L)
In the above formula, M is the molar concentration of monovalent positive ions, G + C is the GC content (%), F is the formamide concentration (%), and L is the length of the probe (base pair base pair: bp) . Also, Tm decreases by about 0.5 to 1.4 degrees due to a 1% mismatch.

  Therefore, by raising the temperature of the solution 11 in the reaction vessel 12 to a temperature higher than the deenergizing temperature Tm, even if the probe P and the target T have undergone self-hybridization, the binding is reliably separated.

Subsequently, while keeping the temperature of the solution 11 in the reaction vessel 12 constant at a temperature higher than the deenergizing temperature Tm (period s3), the power supply 14 is controlled to apply a high-frequency AC voltage as shown in FIG. . As the high-frequency voltage, an electric field of about 1 × 10 ⁶ V / m and about 1 MHz is preferably applied to the solution 11 in the reaction vessel 12 (Masao Washizu and Osamu Kurosawa: “Electrostatic Manipulation of DNA in Microfabricated Structures”, IEEE Transaction on Industrial Application Vol.26, No.26, p.1165-1172 (1990)).

  By applying the high-frequency AC voltage in this way, as shown in FIG. 6, the probe P and the target T expand along the lines of electric force (X1 direction in the figure), and further, the target T becomes the positive electrode 13-1. It moves in the direction toward the side (that is, the direction toward the probe P (direction X1 in the figure)).

  In this way, the probe P and the target T are extended by forming an ion cloud by the phosphate ion (negative charge) forming the skeleton of the nucleotide chain and the hydrogen atom (positive charge) obtained by ionizing water in the vicinity thereof. It is considered that the polarization vector (dipole) generated by these negative and positive charges is directed in one direction as a whole by application of a high frequency high voltage, and as a result, the nucleotide chain is elongated. In addition, when an inhomogeneous electric field in which electric lines of force are concentrated in part is applied, the nucleotide chain moves toward the site where electric lines of force concentrate (Seiichi Suzuki, Takeshi Yamanashi, Shin-ichi Tazawa, Osamu Kurosawa and Masao Washizu: “Quantitative analysis on electrostatic orientation of DNA in stationary AC electric field using fluorescence anisotropy”, IEEE Transaction on Industrial Applications, Vol. 34, No. 1, P75-83 (1998)).

  That is, in the hybridization device 10, the electric lines of force are generated in the direction toward the probe P, and the pair of electrodes 13 is configured so that the density of the electric lines of force increases as the probe P is approached. The probe P and the target T extend in the same direction, and the target T is electrophoresed toward the probe P.

  When the state in which the high-frequency electric field is applied at a temperature higher than the deenergizing temperature Tm is maintained for a certain period of time or longer, the probe P and the target T are sufficiently expanded as shown in FIG. Become. Note that the high-frequency electric field may not be applied after the temperature becomes higher than the deenergizing temperature Tm, but may be applied when the temperature rises during the period s2.

  Subsequently, the heater / cooler 16 is controlled to gradually lower the temperature of the solution 11 in the reaction vessel 12 (period s4). At this time, the power supply 14 is controlled to apply a voltage as shown in FIG. 8 between the electrodes 13 in which “high-frequency electric field” and “no electric field” are alternately applied. By applying such an electric field, the probe P and the target T extend and approach when a high-frequency electric field is applied, and hybridization between the probe P and the target T is promoted when no electric field is applied. That is, in order to advance the mutual reaction such as hybridization between the probe P and the target T with certainty, it is considered more efficient to entrust the Brownian motion of the substance exclusively than the state where the electric field is applied. It is done. However, the nucleotide chain may cause self-hybridization if no high frequency electric field is applied. For this reason, in the hybridization apparatus 10, after the probe P and the target T are extended to rise to the deenergizing temperature Tm, the application state of the high frequency electric field and the non-application state of the electric field are alternately repeated, and the extension and the interaction are performed. Encourage both.

Subsequently, when the temperature of the solution 11 in the reaction vessel 12 reaches the annealing temperature Ta ₁ of a certain probe P, the temperature of the solution 11 in the reaction vessel 12 is held at Ta ₁ for a certain time (period s5). Also at this time, a voltage as shown in FIG. 8 in which “high frequency electric field” and “no electric field” are applied alternately is applied between the electrodes 13. By holding the temperature at the annealing temperature Ta for a certain time as described above, if the base sequences of the probe P and the target T are in a complementary relationship, only the binding between the two is surely performed.

  The annealing temperature is the following temperature. In other words, when DNA is raised above Tm in an aqueous solution, it is separated into ss-DNA, but when the aqueous solution is slowly lowered in temperature and kept slightly lower than Tm, ss-DNA recombines and becomes biologically active. Forms ds-DNA with it. This is called DNA annealing, and the temperature slightly lower than the Tm set at this time is the annealing temperature. The annealing temperature is not particularly limited to a specific single point temperature.

  Subsequently, the temperature of the solution 11 in the reaction vessel 12 is gradually decreased (period s6), and when the annealing temperature of each probe is reached, the temperature of the solution 11 in the reaction vessel 12 is similarly held for a certain period of time (period). s7).

Subsequently, the temperature of the solution 11 in the reaction vessel 12 is gradually lowered to room temperature or Tm or less.
When it reaches a temperature Ti that is neither Tm nor Ta and contributes to the biochemical reaction (period s8), the power supply 14 is controlled to repeat “high frequency electric field”, “no electric field”, and “negative pulse electric field” continuously. A voltage as shown in FIG. 9 is applied between the electrodes 13. When a minus side pulse electric field is applied, the floating target T is electrophoresed in a direction opposite to the direction toward the probe P (X2 direction shown in FIG. 10) as shown in FIG. Therefore, when a high-frequency electric field, a non-electric field, and a negative pulse electric field are continuously applied repeatedly, the target remaining in the solution 11 can be pulled away from the probe without being hybridized. .

  Then, after applying the electric field as shown in FIG. 9 for a certain time and sufficiently separating the target T where no hybridization has occurred from the probe P (period s9), “no electric field” and “minus side pulse electric field” A voltage as shown in FIG. As a result, the target does not extend but is separated from the probe at a high speed, and thereafter unnecessary hybridization does not occur.

  As described above, in the hybridization apparatus 10 to which the present invention is applied, the temperature of the solution is made higher than the highest detent temperature (melting temperature) Tm among the plurality of probes, an alternating electric field is applied, and then the solution is gradually added. Reduce the temperature. As a result, the bonds between all target nucleotide chains (targets) and all detection nucleotide chains (probes) are released and stretched in the direction of the electric field lines of the AC electric field. It becomes.

  Therefore, the hybridization apparatus 10 can cause hybridization at high speed and reliably.

The block diagram of the hybridization apparatus to which this invention was applied is shown. It is a figure which shows the electric lines of force of the electric field applied by an electrode. It is a figure which shows the temperature of the solution in the reaction container in each elapsed time at the time of performing hybridization. It is a figure which shows the state in the reaction container before performing hybridization. It is a wave form diagram of the applied voltage for giving a high frequency electric field. It is a figure which shows the state in the reaction container when a probe and a target expand | extend. It is a figure which shows the state in the reaction container when a target approaches a probe. It is a wave form diagram of an applied voltage when applying a high frequency electric field and a no electric field alternately. It is a wave form diagram of the applied voltage when giving a high frequency electric field, a no electric field, and a minus pulse electric field. It is a figure which shows the state in the reaction container when the remaining target leaves | separates from a probe. It is a wave form diagram of an applied voltage when giving a no electric field and a minus pulse electric field.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 Hybridization apparatus, 11 Solution, 12 Reaction container, 13 Electrode, 14 Power supply, 15 Temperature sensor, 16 Heater / cooler, 17 Controller

Claims (2)

A reaction vessel having a space for storing a solution in which a target nucleotide chain is suspended, and one end of a plurality of probes, which are detection nucleotide chains, fixed to a surface forming the inside of the space,
An anode formed on the outer surface of the reaction vessel and on the same side as the surface to which one end of the probe is fixed, comprising a plate-like member provided in contact with the reaction vessel;
A plate-like member provided so as to be in contact with the reaction vessel, formed on the outer surface of the reaction vessel facing the surface to which one end of the probe is fixed, and a cathode having a larger surface area than the anode;
An AC power source for applying an AC voltage between the anode and the cathode;
A temperature adjusting means for adjusting the temperature of the solution stored on the outer surface of the reaction vessel and other than the surface on which the anode and the cathode are provided, and stored in the reaction vessel;
The solution in the reaction vessel is heated until reaching a temperature higher than that of the probe having the highest deactivation temperature among the probes that are nucleotide chains for detection, and the temperature of the solution is maintained at the reached temperature. In this state, an electric field of about 1 × 10 ⁶ V / m and 1 MHz was generated between the anode and the cathode by applying a voltage between the anode and the cathode. A self-hybridization separation process in which an electric field is applied to the solution by maintaining the electric field for a predetermined time to separate the binding between the probe and the target causing self-hybridization, and after the separation of the self-hybridization, While cooling the solution in the reaction vessel, a voltage is applied between the anode and the cathode, and the anode and the upper A first reaction promoting treatment for promoting extension and hybridization between a probe and a target, which is a nucleotide chain serving as a target, by alternately repeating a state in which no voltage is applied between the cathode and the cathode; When the temperature of the solution in the reaction vessel has reached the annealing temperature of an arbitrary probe after completion of the reaction promotion treatment, a state in which a voltage is applied between the anode and the cathode, and the anode and the above A process for promoting hybridization between an arbitrary probe and a target by continuing the annealing temperature state for a predetermined time while alternately repeating a state in which no voltage is applied between the cathode and the cathode is included in the reaction vessel. After the execution of the second reaction promotion process executed for all the probes and the second reaction promotion process, the reaction volume A state in which a voltage is applied between the anode and the cathode after the temperature of the solution stored therein is cooled to a temperature that does not contribute to a biochemical reaction lower than the annealing temperature of all probes or a room temperature; and By alternately repeating a state in which no voltage is applied between the anode and the cathode and a state in which a voltage generating a negative pulse electric field is applied between the anode and the cathode, an unreacted target is removed from the probe. A first target separation process in which the probe is moved to a surface opposite to the surface on which one end of the probe in the reaction vessel is fixed; and after the first target separation process, the anode and the cathode By alternately repeating a state in which no voltage is applied between and a state in which a voltage generating a negative pulse electric field is applied between the anode and the cathode, unreacted The target is pulled away from the probe and moved to a surface opposite to the surface on which one end of the probe in the reaction vessel is fixed to perform a second target separation process for completely removing the unreacted target. And a controller for controlling the AC power source and the temperature adjusting means. It has a space to store the solution in which the target nucleotide chain floats until it reaches a temperature higher than that of the probe having the highest deactivation temperature among the probes that are the nucleotide chains for detection. The reaction vessel is heated in a state where one end of each probe, which is a nucleotide chain for use, is fixed to the surface forming the space, and the temperature of the solution is maintained at the reached temperature. An anode formed on the outer surface of the reaction vessel and on the same side as the surface to which one end of the probe is fixed, and is provided in contact with the reaction vessel. A cathode having a surface area larger than that of the anode, the plate being formed on the outer surface of the reaction vessel facing the surface to which one end of the probe is fixed. The solution by the application of a voltage, the 1 × 10 6 ^{V /} m between the anode and the cathode to generate an electric field of about 1 MHz, to maintain the electric field generated by a predetermined time during the A self-hybridization separation step of separating the binding of the probe and the target causing self-hybridization by applying an electric field to
After separation of the self-hybridization, while cooling the solution in the reaction vessel, a state in which a voltage is applied between the anode and the cathode, and a state in which no voltage is applied between the anode and the cathode A first reaction promoting step for promoting extension and hybridization between the probe and the target, which is a target nucleotide chain, by alternately repeating
When the temperature of the solution in the reaction vessel reaches the annealing temperature of an arbitrary probe after the first reaction promotion step is finished, a state in which a voltage is applied between the anode and the cathode; A process for promoting hybridization between an arbitrary probe and a target by continuing an annealing temperature state for a predetermined time while alternately repeating a state in which no voltage is applied between the anode and the cathode. A second reaction accelerating step to be performed on all the probes contained therein;
After the execution of the second reaction promoting step, the temperature of the solution stored in the reaction vessel is cooled to a temperature that does not contribute to a biochemical reaction lower than the annealing temperature of all probes or room temperature, and then the anode and A state in which a voltage is applied between the cathode, a state in which no voltage is applied between the anode and the cathode, and a state in which a voltage generating a negative pulse electric field is applied between the anode and the cathode; The first target separation step in which the unreacted target is separated from the probe by alternately repeating the above and moved to the surface opposite to the surface on which one end of the probe in the reaction vessel is fixed;
After execution of the first target separation step, a state where no voltage is applied between the anode and the cathode and a state where a voltage generating a negative pulse electric field is applied between the anode and the cathode alternately By repeating the above, the unreacted target is separated from the probe and moved to the surface opposite to the surface on which one end of the probe in the reaction container is fixed, thereby completely removing the unreacted target. A hybridization method comprising a target separation step.

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