JP2006055025A - Method and vessel for hybridization utilizing bubble - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a vessel for hybridization designed to efficiently stir a micro reaction liquid and improve the hybridization efficiency of a target DNA for a probe DNA immobilized on the substrate surface in measuring a probe carrier. <P>SOLUTION: The method is to stir a reaction liquid in a micro reaction vessel and is carried out as follows. Micro heaters provided in the micro reaction vessel are continuously pulse-heated and the hybridization is carried out while stirring the reaction liquid by expansion and condensation of produced bubbles. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a hybridization reaction method utilizing expansion and condensation of bubbles and a container therefor.

  For example, a substance that specifically binds to a target nucleic acid having a specific base sequence, a so-called probe, can be used to quickly and accurately determine the base sequence of a nucleic acid, detect a target nucleic acid in a sample, and identify various bacteria. A method has been proposed in which a probe array substrate in which a plurality of types of probes are arranged in an array on a solid phase is used to simultaneously evaluate specific binding ability for a plurality of types of probes. A probe carrier, also called a probe array, is a glass substrate, plastic substrate, membrane, etc., on which a probe consisting of several tens to one thousand different types of DNA fragments, etc., is aligned and fixed at high density as spots. is there.

  In recent years, research on detection and quantification of target substances using such probe arrays has been energetically performed. For example, Patent Document 1 discloses a probe array preparation method by sequential DNA extension reaction on a solid phase carrier using photolithography, and Patent Document 2 discloses a probe array preparation method for supplying DNA onto a membrane using a capillary. However, Patent Document 3 discloses a method for preparing a probe array in which a plurality of DNAs are solid-phase synthesized using a piezo-jet nozzle. Patent Document 4 attaches a liquid containing a probe as a droplet to a solid phase using an inkjet head. A probe array fabrication method is described.

  The probe array prepared by the above method detects the presence or absence of the binding between the probe on the probe array and the target substance as a signal after the reaction step (hybridization) with the target substance having an unknown sequence. Thus, an unknown sequence contained in the target substance can be identified or quantified.

In the hybridization reaction, a reaction solution containing a target substance that has been previously labeled with a fluorescent label is dropped on a probe array consisting of a glass plate to which the probe is fixed, and a cover glass is placed on the probe array and left in a constant temperature bath for a certain period of time. Is done in the way. Although some automated containers have appeared, manual (manual) hybridization is still widely performed because the containers are expensive. After that, the probe array is taken out, washed with a cleaning solution or the like, the fluorescent substance used for labeling is excited by a detector, and the target substance that hybridizes with the probe can be identified by reading the fluorescence. .
US Pat. No. 5,424,1865 WO95 / 35505 pamphlet European Patent No. 0703825 Japanese Patent Laid-Open No. 11-187900

  A DNA chip or a DNA microarray must contain several to several tens of μl of a sample containing target DNA dropped on a probe carrier, covered with a cover glass, and placed for several hours. In order to obtain a reliable hybridization result, it is necessary to be able to approach the probe DNA on the probe carrier in a state where the target DNA can be hybridized. However, since the amount of the liquid is very small, it is not easy to stir the liquid, and when it is not stirred, it takes 18 to 24 hours to complete the hybridization. Even with a commercially available hybridization device having a stirring function, hybridization is possible. It takes about 4 hours to complete. Furthermore, a commercially available hybridization instrument having a stirring function has a drawback that it is expensive due to its complicated mechanism.

  Therefore, an object of the present invention is to provide a low-cost, simple, and versatile device that can efficiently agitate a reaction solution in a micro reaction vessel to increase hybridization efficiency and obtain a signal with high quantitative and reproducibility. Hybridization methods and containers are provided.

  The above problems have been solved by the present invention described below.

  (1) A minute reaction vessel provided with at least one minute heater was prepared.

  (2) In the hybridization reaction between the target substance and the probe-immobilized carrier on which the probe capable of specifically binding to the target substance is immobilized, at least one or more micro heaters are pulse-heated, and the generated bubbles are expanded and condensed. By stirring the reaction solution and increasing the hybridization efficiency, it became possible to shorten the reaction time, increase the detection sensitivity, and reduce the variation (standard deviation) in detection sensitivity.

  According to the method of the present invention, the reaction solution in the micro reaction vessel can be efficiently stirred as in the case where the target DNA is hybridized to the probe carrier. In particular, when the target DNA is hybridized to the probe carrier, a stable hybridization result can be obtained in a short time.

  The present invention is a method of stirring a reaction liquid in a micro reaction vessel, in which a micro heater provided in the micro reaction vessel is continuously pulse-heated, and the reaction liquid is stirred by expansion and condensation of generated bubbles. However, the method is characterized by being a method and a container for performing hybridization.

In the present invention, the micro reaction vessel is, for example, a probe carrier hybridization vessel. However, the micro reaction container is not limited to the hybridization container. The micro heater provided in the upper part of the micro reaction vessel has, for example, a thickness of 0.1 to 1 μm, a size of 15 μm × 15 μm to 50 μm × 50 μm, and 3 × 10 ^{4 to} 3 × 10 ⁵ pieces / cm ² . Arranged in a range. In the case of a hybridization container, the microreaction container serves as a spacer between two opposing plates (for example, the upper part of the microreaction container provided with a microheater and a slide glass), and the reaction solution is placed between the plates. It consists of a spacer member (for example, an O-ring) that can be injected. In this case, the internal thickness of the micro reaction vessel (that is, the thickness of the spacer member) is, for example, in the range of 5 to 100 μm.

  At least one of the two plates constituting the micro reaction container is a probe carrier. The reaction solution is a hybridization solution containing target DNA.

  The probe carrier is not particularly limited as long as it does not hinder the detection of the detection substance (target substance) using the probe-fixing substrate obtained by fixing the probe. Examples thereof include materials or polymer materials.

  Specifically, when an inorganic material is used for the base material, in order to introduce a basic group, the base material surface treated with a silane coupling agent having an amino group is preferable. In this case, materials that can be efficiently treated with a silane coupling agent, particularly quartz, glass, silica, alumina, talc, clay, aluminum, aluminum hydroxide, iron, mica, etc. are preferred, but titanium oxide, Oxides such as zinc white and iron oxide can also be used. In view of detection of a target substance and versatility as a material, alkali-free glass or a quartz substrate material that does not contain an alkali component is particularly preferable. Examples of the silane coupling agent having an amino group include N-β (aminoethyl) γ-aminopropyltrialkoxysilane, N-β (aminoethyl) γ-aminopropylmethyldialkoxysilane, γ-aminopropyltrialkoxysilane, Examples thereof include γ-aminopropylmethyl dialkoxysilane. The alkoxysilyl group is preferably a methoxysilyl group or an ethoxysilyl group that can be rapidly hydrolyzed.

  The polymer material is preferably a polymer material having a basic group such as an amino group or a polymer material into which a basic group can be easily introduced. For example, there are a method of using a polyamide having an amino group at the terminal, a method of copolymerizing a substance having a protected amino group and a vinyl group, and removing the protecting group.

  Note that the silane coupling agent in the present invention refers to a compound having both an organic functional group capable of reacting with an organic compound such as a resin and a portion capable of binding to an inorganic compound such as glass via a siloxane bond.

  Further, the shape of the base material is not limited, but if a DNA chip is taken as an example, a substrate shape is preferable from the viewpoint of versatility of detection methods and containers. Furthermore, the substrate material is preferably a substrate material having high surface smoothness, and specifically, a substrate having a size of about 1 inch × 3 inches and a thickness of about 0.7 to 1.5 mm is preferable.

When the surface of the substrate is treated with a silane coupling agent, it is preferable to wash the substrate in advance. As cleaning methods, there are many known methods such as cleaning with water, cleaning with chemicals, cleaning with plasma, cleaning with UV ozone, etc., but cleaning methods with chemicals are simple and uniform cleaning methods. Is appropriate. Although a suitable cleaning method varies depending on the type of the substrate, for example, when glass is used as the substrate, the substrate surface is sufficiently cleaned using a sodium hydroxide aqueous solution of a predetermined concentration and adhered onto the substrate. A method for removing dirt is mentioned. Specifically, a 1M sodium hydroxide aqueous solution heated to about 60 ° C. is prepared, and wiping of the substrate surface in the aqueous solution or brushing while showering the aqueous solution ensures reliable adhesion on the substrate. To remove. After removing the dirt, thoroughly wash away excess sodium hydroxide with water. Finally perform water removal by methods such as blown with an inert gas such as N _2.

  As a method for coating the silane coupling agent, a dipping method (dipping method), a spin coating method, a spray coating method, or the like can be used. In particular, a dipping method that allows simple and uniform treatment is preferred. It is preferable to treat the substrate by immersing the cleaned substrate in an aqueous solution of silane coupling agent dissolved in water having a concentration of 0.1 to 2.0 wt%, and washing away the solution containing excess silane coupling agent after completion of the reaction. However, the coating method is not particularly limited.

  Moreover, it is preferable to remove an excess silane coupling agent from a base material, and to dry at the temperature of about 100-120 degreeC.

  Probes used in the present invention include biopolymers such as proteins (including complex proteins), nucleic acids, sugar chains (including complex carbohydrates), lipids (including complex lipids), and the like. Specific examples include enzymes, hormones, pheromones, antibodies, antigens, haptens, peptides, synthetic peptides, DNA, synthetic DNA, RNA, synthetic RNA, PNA, synthetic PNA, ganglioside, and lectin. The amount of the probe contained in the medium is, for example, in the case of a nucleic acid probe, considering probe stability, for example, a nucleic acid probe of 2 mer to 500 mer, particularly 2 mer to 80 mer, 0.05 to 500 μM, In particular, it is preferable to adjust the concentration to 0.5 to 50 μM.

  When introducing a thiol group into a probe, for example, when a DNA to be synthesized automatically is used as a probe, a thiol modifier (manufactured by Glen Research) should be used for synthesis with an automatic DNA synthesizer. Can do. In addition, if a thiol group can be introduce | transduced efficiently, it will not specifically limit.

  In applying the probe, an aqueous liquid in which the probe is dissolved or dispersed in an aqueous medium is spotted on a substrate having a basic group by an inkjet method, a pin method, a pin & ring method, or the like.

  Regarding the spotting method, among the above-described methods, the inkjet method is a preferred spotting method because high-density and accurate spotting can be performed. Inkjet method is a method in which a solvent containing a probe is placed in a very thin nozzle, and the vicinity of the nozzle tip is momentarily pressurized or heated, and the solvent containing a very small amount of probe is ejected from the nozzle tip accurately. Is made to fly and adhere to the substrate surface. In the spotting method based on the ink jet method, the components contained in the probe medium do not substantially affect the probe when ejected from the ink jet head as the probe medium, and are formed on the substrate using the ink jet head. There is no particular limitation as long as it satisfies a medium composition that can be normally ejected. For example, when the inkjet head is a bubble jet (registered trademark) head having a mechanism for applying thermal energy to the medium and ejecting it, the liquid containing glycerin, thiodiglycol, isopropyl alcohol, and acetylene alcohol is a component contained in the probe medium Is preferable. More specifically, a liquid containing glycerin 5 to 10 wt%, thiodiglycol 5 to 10 wt%, and acetylene alcohol 0.5 to 1 wt% is suitably used as the probe medium. When the inkjet head is a piezo jet head that ejects a medium using a piezoelectric element, a liquid containing ethylene glycol and isopropyl alcohol is preferable as a component contained in the probe medium. More specifically, a liquid containing 5 to 10 wt% ethylene glycol and 0.5 to 2 wt% isopropyl alcohol is preferably used as the probe medium.

  When the probe medium thus obtained is ejected from the ink jet head and deposited on the substrate, the spot shape is circular and the ejected range does not widen. Even when the probe medium is spotted at a high density, the connection with adjacent spots can be effectively suppressed. The characteristics of the probe medium of the present invention are not limited to the above.

  In addition, an aqueous liquid in which a silane coupling agent having a probe and a basic group is dissolved or dispersed in an aqueous medium is brought into contact with the substrate surface by a method such as an ink jet method or a pin method, so that the basic group to the substrate is contacted. And the probe may be fixed at the same time.

  Further, in order to reduce the drying of the spot probe, a substance having a high boiling point may be added to an aqueous liquid in which the probe is dissolved or dispersed. The substance having a high boiling point is preferably a substance that can be dissolved in an aqueous solution in which the probe is dissolved or dispersed and that is not highly viscous. Such materials include glycerin, ethylene glycol, diethylene glycol, thiodiglycol, dimethyl sulfoxide and low molecular weight hydrophilic polymers. Examples of the hydrophilic polymer include polyvinyl alcohol, polyvinyl pyrrolidone, paogen, carboxymethyl cellulose, hydroxyethyl cellulose, dextran, pullulan, polyacrylamide, polyethylene glycol, and sodium polyacrylate. More preferably, ethylene glycol or diethylene glycol is used as the high boiling point substance. The concentration of the high-boiling substance is preferably in the range of 0.1 to 10% by volume in the probe aqueous solution. Moreover, the solid phase carrier after application of the probe may be in an environment having a humidity of 90% or more and a temperature range of 20 to 50 ° C.

  After spotting, it is preferable to remove excess probe by washing. Although depending on the type of probe, when a single-stranded DNA probe into which a thiol group is introduced is used, the probe is fixed within 1 minute, but it is preferable to remove it after standing for 10 minutes or longer.

  The probe-immobilized substrate thus obtained is suitable as a probe-immobilized carrier for detecting a target substance.

  Here, for example, for the purpose of improving the detection accuracy (S / N ratio) when detecting a target substance using this probe-immobilized carrier, Blocking may be performed so that the probe non-binding portion on the material does not bind to the target substance or the like contained in the specimen (sample). The blocking is performed, for example, by immersing the base material in a 1-2% bovine serum albumin aqueous solution for about 2 hours. However, in view of the effect of preventing the labeling substance from adsorbing to other than the probe fixing site on the substrate, an aqueous bovine serum albumin solution is preferable. This blocking step may be performed as necessary. For example, the sample is supplied to the probe-fixing base material in a limited manner for each spot, so that the sample adheres to a portion other than the spot substantially. If not, you do not have to. When a sample is attached to a site other than the spot, the sample differs depending on the material used as the base material and the type of the silane coupling agent. Further, when the base material is glass, quartz or the like, the blocking operation is not necessary when spotting a probe medium including a substance such as a silane coupling agent having a basic group and a probe having a thiol group.

  The probe fixing base material thus produced may be configured to have, for example, a plurality of spots including the same probe, or a plurality of spots each including a different type of probe, depending on the application. It may be configured. The type, quantity, and arrangement of the probes can be changed as necessary. The probe-immobilized base material on which the probes are arranged at high density by such a method is then used for detection of the target substance, identification of the target substance base sequence, and the like.

  For example, when used to detect a single-stranded nucleic acid whose target sequence may be contained in a sample and whose target sequence is a known target substance, it is complementary to the base sequence of the single-stranded nucleic acid of the target substance. A single-stranded nucleic acid having a simple base sequence is used as a probe, and a probe-fixing substrate in which a plurality of spots including probes are arranged on a solid phase is prepared, and a detection substance is applied to each spot on the probe-fixing substrate. After the sample is applied and placed under conditions such that the single-stranded nucleic acid of the target substance and the probe hybridize, the presence or absence of the hybrid in each spot is detected, for example, by detection by fluorescence, radio waves, magnetic force, etc. Detect with a known method. Thereby, the presence or absence of the target substance in the sample can be detected.

  In addition, when using to identify the base sequence of a single-stranded nucleic acid of a target substance contained in a sample, multiple candidates for the base sequence of a single-stranded nucleic acid of the target substance are set, and Then, single-stranded nucleic acids each having a complementary base sequence are spotted on a substrate as a probe. Next, after supplying a sample to each spot and placing under conditions such that the single-stranded nucleic acid of the target substance and the probe hybridize, the presence or absence of the hybrid in each spot is determined by a known method such as fluorescence detection. To detect. Thereby, the base sequence of the single-stranded nucleic acid which is a target substance can be specified. Further, as other uses of the probe-immobilizing substrate according to the present invention, for example, application to screening of a specific base sequence recognized by a DNA-binding protein and screening of a chemical substance having a property of binding to DNA can be considered.

  Hybridization is preferably performed by applying an aqueous solution in which a labeled sample nucleic acid fragment is dissolved or dispersed on the DNA chip prepared above. Hybridization is preferably carried out in the temperature range of room temperature to 70 ° C. and in the range of 2 to 20 hours. At this time, at least one or more micro heaters provided in the upper part of the micro reaction vessel are continuously pulse-heated, and the reaction liquid is stirred by expansion and condensation of the generated bubbles. The method for heating is, for example, a heating pulse width of 0.5 to 3.5 μsec, an applied voltage of 6 to 15 V, and a heating pulse frequency of 1 to 10 KHz.

  After completion of the hybridization, it is preferable to remove the unreacted sample nucleic acid fragment by washing with a mixed solution of a surfactant and a buffer solution. As the surfactant, sodium dodecyl sulfate (SDS) is preferably used. As the buffer solution, a citrate buffer solution, a phosphate buffer solution, a borate buffer solution, a Tris buffer solution, a Good buffer solution, and the like can be used, and it is particularly preferable to use a citrate buffer solution.

  FIG. 1 is a schematic view of a hybrid container for carrying out the method of the present invention. The upper figure is a plan view and the lower figure is a side view. The hybrid container 1 includes a slide glass 5 (DNA chip), a cover plate 10 provided with at least one or more micro heaters 6 provided facing the slide glass 5, and a space between the slide glass 5 and the cover plate 10. It consists of an O-ring 4 that is a spacer member for maintaining, a liquid injection port 3 to the reaction vessel 7, a discharge port 2, a heater 8 for adjusting the temperature of the reaction liquid, and a fixing screw 9 for fixing the cover plate. The range is about 20 mm × 60 mm, the thickness is about 0.12 mm, and the volume is about 150 μl.

  A predetermined amount (150 μl) of the reaction solution is injected into the reaction vessel 7 from the injection port 3. At least one or more micro heaters 6 are disposed over the entire surface of the cover plate 10 as shown in FIG. After injecting the reaction solution, at least one or more micro heaters 6 are sequentially heated to generate bubbles on the micro heater 6 and expand them, and then stop at least one or more micro heaters 6 from being heated sequentially. The reaction liquid moves (flows) as the bubbles expanded on the minute heater 6 condense. The reaction solution is stirred as the reaction solution moves.

  This state is shown in FIG. FIG. 2 shows a state where bubbles generated by heating the minute heater 61) are expanding, and a state where bubbles are condensed because the minute heaters 62) and 3) are not heated. The reaction liquid moves (flows) from left to right by expansion and condensation.

  In FIG. 2, the minute heaters 6 are sequentially heated to cause a liquid movement (flow) in one direction and stir the reaction liquid. For example, a plurality of minute heaters 6 may be heated simultaneously in each area or adjacent to each other. Stirring can also be performed by generating bubbles by alternately heating the minute heaters 6 that are in contact with each other and moving (flowing) the reaction liquid by expansion and condensation.

  During stirring of the reaction solution by bubble expansion and condensation, the temperature of the reaction solution can be adjusted using the heater 8 to a temperature suitable for the reaction. In the case of hybridization, the temperature of the reaction solution is, for example, from room temperature to 90 ° C. Moreover, reaction time can be suitably determined according to the kind of reaction. However, according to the method of the present invention, even a very small amount of reaction solution can be stirred more efficiently, so that the reaction time can be shortened.

  After completion of the reaction, the reaction solution is discharged from the discharge port 2, the reaction vessel 7 is appropriately washed and dried, and kept at a constant temperature by the heater 8. When the slide glass 5 is a probe carrier, the slide glass 5 is recovered and subjected to a hybridization detection operation (for example, fluorescence analysis for detecting hybridized DNA).

  Although FIG. 1 shows one reaction container, the container used in the method of the present invention is a unit (waste liquid) for supplying or recovering at least one reaction container and a reaction liquid and a washing liquid to the reaction container. It is also possible to provide a composite apparatus including a tank, a cleaning liquid tank, a liquid feeding pipe and a pump.

  Hereinafter, the present invention will be described in more detail. Such embodiments are not limited to those illustrated here.

(1) Probe synthesis A single-stranded DNA probe was used as a probe capable of specifically binding to a target substance. A single-stranded nucleic acid of SEQ ID NO: 1 was synthesized using an automatic DNA synthesizer. Note that a mercapto (SH) group was introduced into the 5 ′ end of the single-stranded DNA end of SEQ ID NO: 1 at the time of synthesis using an automatic DNA synthesizer. Subsequently, deprotection was performed, the DNA was recovered, purified by high performance liquid chromatography, and used in the following experiments.
SEQ ID NO: 1 5 'ACTGGCCGTCGTTTTACA 3'
(2) Preparation of carrier Maleimide groups were introduced on the surface of six carriers in the same manner as described in (0087) Examples 1 to (0088) of Patent Document 4.

(3) Probe ejection by BJ printer and binding to carrier As spotting solutions used for probe spotting, 7.5% by mass of glycerin, 7.5% by mass of urea, 7.5% by mass of thiodiglycol, and acetylene alcohol (Product name: Acetylenol EH; manufactured by Kawaken Fine Chemical Co., Ltd.) An aqueous solution containing 1% by mass is prepared, and the solution containing the single-stranded DNA (1) (single-stranded DNA concentration is about 400 mg / ml). In addition to a certain TE solution (10 mM Tris-HCl (pH 8) / 1 mM EDTA aqueous solution), the final concentration of single-stranded DNA was adjusted to 8 μM. The surface tension of the spotting solution containing this probe is in the range of 20 to 50 dyne / cm, and the viscosity is 1.2 to 2.0 cps (E-type viscometer: manufactured by Tokyo Keiki Co., Ltd.).

  The spotting solution containing the probe was filled in an ink tank for a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) and mounted on a bubble jet (registered trademark) head. In addition, the bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here is modified so that printing on a flat plate is possible. Next, the carrier prepared in (2) above was mounted on this printer, and a spotting solution containing the probe was spotted on the carrier to produce a probe carrier. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the carrier was 1.2 to 1.5 mm. After the spotting, the carrier was observed with a microscope, and it was confirmed that a matrix spot array was formed on the carrier surface. After completion of the reaction between the maleimide group and the thiol group, the probe carrier was washed with 1 M NaCl / 50 mM phosphate buffer (pH 7.0), and the liquid containing the probe on the surface of the probe carrier was completely washed away. Six prepared probe carriers were stored as they were immersed in 1M NaCl / 50 mM phosphate buffer (pH 7.0).

(4) Blocking treatment Six probe carriers prepared in the above (3) were immersed in a 2% bovine serum albumin aqueous solution and left for 2 hours to perform a blocking treatment.

(5) Hybridization treatment A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 1 described in (1) above is synthesized by an automatic DNA synthesizer, and rhodamine is added to the 5 ′ end. A single-stranded DNA probe labeled by binding was obtained. This labeled single-stranded DNA was dissolved in 1 M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM to prepare a hybridization reaction solution.

  Six probe carriers after completion of the blocking treatment of (4) above were prepared, and the six probe carriers were placed in a micro reaction vessel and placed on a heater (Peltier element).

  A total of six micro reaction containers were filled with the hybridization reaction solution, and the temperature of the heater (Peltier element) was kept at 45 degrees to perform the hybridization reaction for 2 hours. In the example (with stirring), the microheater installed on the top of the three microreaction vessels is continuously pulse-heated, and the reaction solution is moved (flowed) by the expansion and condensation of the generated bubbles. Was stirred. The comparative example (without stirring) was performed in the same manner as in the example except that the remaining three micro reaction vessels were not heated by the three micro heaters.

  After completion of the hybridization reaction, washing was performed with 1M NaCl / 50 mM phosphate buffer (pH 7.0) to wash away the labeled single-stranded DNA that had not undergone the hybridization reaction. Then, after removing excess salt with pure water, each carrier was dried by nitrogen blowing. Next, the fluorescence intensity of the spots on each carrier was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.

(6) Results The fluorescence scanner evaluation results in (5) above were analyzed, and the fluorescence intensity of each of the three carriers in the comparative example (without stirring) was averaged. The fluorescence intensity at 532 nm of the labeled single-stranded DNA probe subjected to the hybridization reaction was 8320. The fluorescence intensity of the portion of the probe carrier where no probe was present was 81, and the S / N was about 100.

  On the other hand, when the fluorescence intensities of the three carriers in the examples (with stirring) were averaged, the labeled single-stranded DNA probe that had undergone a hybridization reaction with a perfect match with the probe of SEQ ID NO: 1 on the probe carrier at 532 nm The fluorescence intensity was 16120. The fluorescence intensity of the portion of the probe carrier where no probe was present was 82, and the S / N was about 200. Furthermore, the fluorescence intensity variation (standard deviation) of each probe was about 30% smaller than that of the comparative example (without stirring).

A hybrid container for carrying out the present invention. The imaginary view of the stirring by expansion | swelling of the bubble generated from the micro heater in the method of this invention, and condensation.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Hybrid container 2 Outlet 3 Inlet 4 O-ring 5 Slide glass 6 Micro heater 7 Reaction container 8 Heater (for temperature control)
9 Fixing screw

Claims (11)

  A hybridization reaction method comprising heating at least one or more minute heaters provided in a minute reaction vessel and stirring the reaction solution by expansion and condensation of the generated bubbles.   2. The method according to claim 1, wherein fluctuations in bubble expansion and condensation are performed by sequentially or randomly heating at least one or more micro heaters provided in the micro reaction vessel.   The method according to claim 1, wherein the bubble expansion and condensation fluctuations are performed by simultaneously heating at least one or more micro heaters provided in the micro reaction vessel.   2. The method according to claim 1, wherein fluctuations in expansion and condensation of the bubble are performed by changing a heating time of at least one or more micro heaters provided inside the micro reaction vessel.   2. The method according to claim 1, wherein fluctuations in bubble expansion and condensation are performed by changing an applied voltage of at least one or more micro heaters provided inside the micro reaction vessel.   The method according to claim 1, wherein the fluctuation of bubble expansion and condensation is performed by changing a heating frequency of at least one or more micro heaters provided inside the micro reaction vessel.   A probe carrier hybridization container, wherein a plurality of the micro heaters according to claim 1 are provided on an upper surface of the micro reaction container.   8. The hybridization container according to claim 7, wherein the minute heater has a thickness in a range of 0.1 to 1 μm.   9. The hybridization container according to claim 7, wherein a size of the micro heater is in a range of 15 μm × 15 μm to 50 μm × 50 μm. The hybridization container according to any one of claims 7 to 9, wherein the micro heater is arranged in a range of 3 x 10 ^{4 to} 3 x 10 ⁵ pieces / cm ² inside the micro reaction container.   The hybridization container according to claim 7 to 10, wherein the internal thickness of the micro reaction container is in the range of 5 to 100 µm.

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