JP2007114103A - Probe-fixing gel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an efficient reaction between a probe fixed to a gel network structure by probe-fixing gel and a target specimen, the probe-fixing gel being made by fixing the probe in the network structure with spatial flexibility. <P>SOLUTION: The probe and the gel containing a high-molecular compound are prepared and then the high-molecular compound in the gel is removed to obtain the probe-fixing gel. The probe-fixing gel allows an efficient reaction between the probe and the target specimen. This gel can be used also as a member of a DNA micro-array. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a probe-immobilized gel that can efficiently detect a target sample.

  In the late 1990s, microarrays appeared as a technology that promoted transcriptome analysis. Microarrays, also called DNA chips, are DNA molecules arranged at high density on a small substrate made of glass or silicon. By using a microarray, various gene expressions can be observed simultaneously.

DNA microarrays are roughly divided into two types, the Stanford method and the Affymetrix method, depending on the principle of creation. The Stanford DNA microarray is a microarray in which DNA fragments prepared in advance, for example, those amplified by a PCR method from a cDNA-holding plasmid, are spotted at high density on a slide glass (Non-patent Document 1). On the other hand, Affymetrix's microarray is a microarray produced by synthesizing about 20-25mer oligonucleotides on a substrate by combining photolithographic technology and light irradiation chemical synthesis (Non-patent Document 2).

  In recent years, a microarray using a through-hole using a through-hole substrate has been developed as another technique. For example, a flow-through chip manufactured by Metricens Inc. immobilizes nucleic acid as a probe on the surface of a silicon substrate on which minute through holes are regularly arranged, and allows a target nucleic acid sample solution to flow through the through hole. Thus, the nucleic acid probe and the target nucleic acid are hybridized (Patent Document 1).

  Furthermore, as another example of the microarray using the through hole, the present inventor has also developed a fiber microarray although a hollow fiber array is used. This microarray uses a hollow fiber array in which a plurality of hollow fibers are regularly arranged in the fiber axis direction, and a nucleic acid to be a probe is immobilized in each hollow fiber hollow portion of the hollow fiber array, and this hollow fiber array It can be manufactured by slicing the body by a method perpendicular to the fiber axis direction (Patent Document 2).

  On the other hand, as a method for fixing a probe such as a biological substance, a method for fixing a probe to a gel is also known. For example, Patent Document 3 discloses a method of reacting acrolein with an acrylamide monomer and a methylenebisacrylamide, which are gel-forming polymerizable monomers, as an example of a bio-related substance. In this method, the amidation-modified nucleic acid and the acrolein aldehyde residue are Schiff-base bonded, while the vinyl residue on one side of the acrolein is copolymerized with acrylamide to covalently immobilize the nucleic acid on the acrylamide gel. It is a method to do.

  The above-described method for immobilizing a probe on a gel carrier is also applied to microarray technology. For example, an acrylamide gel is coated on a glass substrate, and the gel is held in the hollow portion of the microarray (Patent Document 4, Patent Document 5) in which the nucleic acid probe is fixed to the gel layer, and the above-described fiber type microarray. A microarray (Patent Document 6) is known.

  In the case of a microarray in which the probe is immobilized on a gel, the probe is immobilized on the three-dimensional network structure of the gel, and the probe is immobilized on a two-dimensional surface such as the above-mentioned Stanford microarray or Affymetrix microarray. Compared to a microarray, a three-dimensional space can be effectively used as a field (space) in which a probe reacts.

However, in order to effectively use the gel network as a field where the probe reacts, for example, consider the size of the probe, the size of the molecule that reacts with the probe, and the three-dimensional freedom of the complex. It is necessary to construct a gel network structure.

For example, the pore size of a normal acrylamide gel depends on the monomer concentration and cross-linking ratio to be set, but it is about 1.5 to about under generally used conditions (concentration: 3% to 10%, cross-linking ratio: 5%). The range is 2.0 nm. On the other hand, the molecular size (diameter) of DNA is about 2.0 nm, which is almost the same as the pore size of the gel when it has a double helix structure. Therefore, in the gel, the spatial degree of freedom of nucleic acids is considerably limited.
Science 270,467-470 (1995) Science 251,767-773 (1991) Japanese Patent No. 3488456 JP 2000-245460 A Japanese Unexamined Patent Publication No. 3-47097 Special Table 2003-524150 WO2000-31148 Publication Japanese Patent No. 3510882

  Therefore, an object of the present invention is to provide a probe-immobilized gel in which a probe is immobilized in a gel network structure having a spatial degree of freedom, and to perform an efficient reaction between the probe of the probe-immobilized gel and a target sample. It is to be realized.

  As a result of intensive studies in view of the above-mentioned problems, the present inventors gave a spatial restriction in advance during gel formation, and opened the spatial restriction after the gel formation, so that the vicinity of the probe in the gel structure The present invention has been completed by finding that it has spatial freedom.

  That is, the present invention is a probe-immobilized gel obtained by preparing a gel containing a probe and a polymer compound and then removing the polymer compound from the gel.

  Since the probe is fixed in the gel network structure having a spatial degree of freedom, the probe and the target sample can react efficiently.

  Hereinafter, the present invention will be described sequentially.

  The present invention is “a probe-immobilized gel obtained by preparing a gel containing a probe and a polymer compound and then removing the polymer compound”.

  As a first step for obtaining the gel, a gel containing a probe and a polymer compound is prepared. As used herein, the term “probe” refers to a probe that detects a target substance. If the target substance is a nucleic acid, the probe is a nucleic acid complementary to the nucleic acid, a protein having a binding property to the nucleic acid, or the like. It is. The probe is fixed to the gel by the method described later.

  The “polymer compound” is a compound for forming a space in the vicinity of the probe immobilized gel of the present invention. This high molecular compound is removed by the 2nd process mentioned later. A space is created in the removed part. As the kind of the polymer compound, any substance can be adopted as long as it has an affinity for the gel or its precursor and does not impair the function of the probe. For example, a hydrophilic polymer compound can be employed. Specific examples include polyethylene glycol, polyether, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, and hydroxymethyl cellulose. In addition, proteins, peptides, sugar chains, nucleic acids, complexes thereof, and the like can be used.

  When a nucleic acid is selected as the polymer compound, any of DNA, RNA, PNA and the like can be used as the nucleic acid. In addition, they may be either single-stranded or double-stranded. Furthermore, the chain length is preferably 1 to 2 kb in view of the purpose of providing a space in the gel.

  Moreover, as a high molecular compound, what has a bondability with the said probe is preferable. By having binding properties with the probe, it is possible to provide a space closer to the probe. Further, the bonding force is preferably such that it can be easily dissociated, and hydrogen bonding, electrostatic bonding, and the like are preferable. For example, when the probe is a nucleic acid, a nucleic acid containing a sequence complementary to the nucleic acid can be selected as the polymer compound. By selecting such a polymer compound, a space can be intentionally created in the vicinity of the probe, and removal is facilitated.

  The type of “gel” is not particularly limited, and a physical gel or a chemical gel can be used. If it is a physical gel, agarose, agaropectin, amylose, amylopectin, gum arabic, arabinan, isorikenan, insulin, ethylcellulose, ethylhydroxyethylcellulose, curdlan, casein, carrageenan, carboxymethylcellulose, carboxymethyl starch, callose, agar, xanthan gum, chitin , Guar gum, chitosan, quince seed, crown gall polysaccharide, glycogen, glucomannan, keratan sulfate, red alga starch, yeast mannan, collagen, gelatin, starch, alginate, propylene glycol alginate, gellan gum, schizophyllan, cellulose, elephant palm Mannan, Tamarind Seed Gum, Tunisin, Nigeran, Hydroxypropylcellulose, Pustulan Funolan, decomposed xyloglucan, HM pectin, polyamidine, polyperfluorocarbon sulfonic acid, polymethacrylic acid-polyethylene glycol copolymer, polymethoxyethylene glycol methacrylate, porphyran, methylcellulose, methyl starch, lentinan, locust bean gum, polyvinyl alcohol, poly Sodium acrylate can be used.

  For chemical gels, polyacrylamide gel, polyisopropylacrylamide gel, dimethylacrylamide gel, polyvinylpyrrolidone gel, silk fibroin, keratin protein, 2- (dimethylamino) ethyl methacrylate, 2-dimethylaminoethyl methacrylate gel, HS protein Dextran, hyaluronic acid gel, bisacrylamide methyl ether gel, poly- (2-acrylamido-2-methylpropanesulfonic acid) gel, polyacrylic acid gel, poly- (N, N-acrylic acid dimethylaminoethyl ester) gel, Cross-linked sodium polyacrylate, poly- (N-acryloylaminoethoxyethanol) gel, poly- (N-acryloylaminopropanol) gel, polyacryloxypropane sulfonic acid gel, polyisobutylene / maleic acid copolymer gel , Polyethyleneimine gel, polyethylene oxide / propylene oxide copolymer gel, polyethylene oxide / polyethylene glycol gel, polyethylene-vinyl acetate copolymer gel, polydioxolane gel, polydimethylaminopropylacrylamide gel, poly- (2-hydroxyethyl methacrylate) Poly (2-hydroxyethyl methacrylate) gel, polyvinyl pyridine gel, polyvinyl methyl ether, polypropylene oxide, poly- (N-methylol acrylamide) gel and the like can be used.

  Examples of the method of including the probe and the polymer compound in the gel include a method of including these substances in a gel precursor solution and gelling by cooling or the like if the gel is a physical gel. Here, the probe can be chemically fixed to the gel by chemically modifying the probe and / or the gel precursor in advance. For example, when agarose is selected as the physical gel, the probe is covalently fixed to the gel by exposing an aldehyde group by periodate oxidation and reacting the aldehyde group with an amino group-modified probe. Can do. Moreover, an unsaturated functional group is introduce | transduced into the hydroxyl group of agarose, and a probe can also be fixed to a gel by copolymerization reaction with the probe which introduce | transduced the unsaturated functional group (refer Electrophoresis 1995, 16, p1337-1344). Various known methods can be adopted as the probe fixing method in consideration of the stability of the probe or gel.

  In the case of a chemical gel, a probe and a polymer compound are included in a gel precursor solution such as a monomer and a polymerization initiator, and these compounds can be included in the gel by carrying out a polymerization reaction. Here, if an unsaturated functional group is introduced into the probe in advance, the probe can be covalently bonded to the constituent components of the gel during the polymerization reaction (see JP-A-3-47097).

  In addition, as a method for fixing the probe to the gel, a method in which the probe is fixed to a particle or the like in advance and the particle is included in the gel is also included. In this case, any particles may be used as long as the particles are uniformly dispersed in the gel.

  Next, in the second step, the polymer compound is removed from the gel prepared as described above. Examples of the removal method include a method of removing the polymer compound by natural diffusion using a concentration gradient, a method of driving the polymer compound out of the gel system by heating, and a method of removing the polymer compound from the gel system using electrophoresis. The method of moving is mentioned.

  Moreover, removal becomes easy by decomposing | disassembling a high molecular compound previously. For example, when a nucleic acid is used as the polymer compound, it can be easily removed by decomposing the nucleic acid with a nucleolytic enzyme or the like. When DNA is selected as the probe, RNA is included as a polymer compound, a gel is prepared, and RNA is decomposed with RNase or the like, so that the polymer compound is decomposed without affecting the DNA of the probe. Can be removed. Further, when a protein is used as the polymer compound, it can be easily removed by decomposition with a protease or the like.

  The gel of the present invention is held in a tubular body and can be used as a device for genetic testing (see JP-A-3-47097). For example, a gel precursor solution containing a probe and a polymer compound is introduced into a tubular body, a gelation reaction is performed in the tubular body, and then the polymer compound is removed from the gel, whereby the gel is held in the tubular body. A gel-holding tubular body can be produced.

  The gel of the present invention can also be used as a gel for holding microarray probes. For example, a plurality of gel precursor solutions containing a probe and a polymer compound are dropped on a flat substrate, and a gelation reaction is performed on the substrate. After the gelation reaction, the microarray can be produced by removing the polymer compound from the gel.

  The gel of the present invention can also be suitably used for a microarray in which probes are fixed to the through holes of a substrate having a plurality of through holes (Japanese Patent Laid-Open No. 2000-60554). That is, a gel precursor solution containing a probe and a polymer compound is introduced into the through hole, and a gelation reaction is performed in the through hole. After the gelation reaction, the microarray can be produced by removing the polymer compound from the gel. The removal of the polymer compound is preferably carried out by placing electrodes on the front and back of the microarray and performing electrophoresis.

  Furthermore, the gel of the present invention can be suitably used as a gel for holding a probe of a fiber type microarray developed by a part of the present inventors (Japanese Patent No. 3510882). Specifically, by filling the hollow portion of the hollow fiber with a gel precursor solution containing a probe and a polymer compound, performing a gelation reaction in the hollow portion, and then removing the polymer compound from the gel, the fiber Type microarrays can be manufactured.

  Hereinafter, details of the embodiment will be described in Examples.

<Example 1> Production of nucleic acid microarray
1. Preparation of probe A 5′-terminal vinylated nucleic acid molecule (65mer) consisting of the following base sequence was used as a probe.

Sequence number 1: CCGCCTGCTC CCAGAGGTGT GCCTCAAATT GATGTTACCT TTGATATCGA CGCTAATGGT ATTCT
The probe was synthesized according to the following procedure. First, in order to synthesize a terminal aminated nucleic acid 5′-O-aminohexyl-nucleic acid, an oligonucleotide having the above base sequence was synthesized by an automatic DNA synthesizer using an amidite reagent. Thereafter, in the final stage, aminolink ^™ (manufactured by PE Biosystems) was reacted with the oligonucleotide and then synthesized by deprotection.

2. Preparation of polymer compound (aRNA) RNA was selected as the polymer compound. The RNA was prepared by preparing cDNA from total RNA according to the following procedure, and transcribing it from aRNA.

(1) Preparation of first strand cDNA from total RNA
A solution containing T7 Oligo dT Primer (Ambion # 5710), Universal mouse Total RNA 5 μg (Stratagene) and DEPC-treated water was prepared in a 0.2 ml-PCR tube (Table 1).

上記のRNA sampleを70℃で5分間インキュベーションし、チューブを氷上に置き、遠心後、表２に示す試薬を添加した。 [Table 1]

The above RNA sample was incubated at 70 ° C. for 5 minutes, the tube was placed on ice, centrifuged, and the reagents shown in Table 2 were added.

次にボルテックス^TMで攪拌、遠心後、45℃で2分間インキュベーションし、続いて、SuperScript II Reverse Transcriptase^TM（Invitrogen社製）1μlを静かに加え、45℃で60分間インキュベーションした。この時、チューブ内の総液量は20μlになった。反応終了後、チューブを取り出し、氷上に静置した。 [Table 2]

Next, the mixture was stirred with vortex ^™ , centrifuged, and incubated at 45 ° C. for 2 minutes. Subsequently, 1 μl of SuperScript II Reverse Transcriptase ^™ (Invitrogen) was gently added and incubated at 45 ° C. for 60 minutes. At this time, the total liquid volume in the tube was 20 μl. After completion of the reaction, the tube was taken out and left on ice.

(2) Preparation of Second strand cDNA The tube containing the First strand cDNA sample (20 μl) obtained in (1) was placed on ice, and the reagents shown in Table 3 were added. After the addition, the mixture was gently agitated and incubated at 16 ° C. for 2 hours.

次にT4 DNA polymeraseを2μｌ添加し、さらに16℃で15分間インキュベーションした。反応が終了したら、チューブを取り出し、氷上に静置した。 [Table 3]

Next, 2 μl of T4 DNA polymerase was added and further incubated at 16 ° C. for 15 minutes. When the reaction was completed, the tube was taken out and left on ice.

(3) Purification of Second strand cDNA For purification, QIAquick ^™ PCR Purification Kit (# 28104 manufactured by QIAGEN) was used.

All of the second strand cDNA solution (150 μl) prepared in (2) was transferred to a sterilized tube (1.5 ml volume), and then 750 μl (5 times volume) BufferPB was added, stirred with vortex ^™ and centrifuged.

The QIAquick ^™ spin column was inserted into a 2 ml collection tube, and 450 μl of the second strand cDNA mixture was added to the QIAquick ^™ spin column and centrifuged at 13,000 rpm for 30-60 seconds. The eluate was discarded, and the QIAquick ^™ spin column was again inserted into a collection tube. The remainder (450 μl) of the mixed solution was placed in the QIAquick ^™ spin column, and centrifuged at 13,000 rpm for 30-60 seconds.

The eluate was discarded, and a QIAquick ^™ spin column was again inserted into the collection tube, 700 μl of BufferPE was added, and centrifuged at 13,000 rpm for 30-60 seconds.

The eluate was discarded, and the QIAquick ^™ spin column was again inserted into the collection tube, and centrifuged at 13,000 rpm for 30-60 seconds to completely remove excess BufferPE on the filter.

A QIAquick ^™ spin column was inserted into a new 1.5 ml tube, 20 μl of DEPC-treated water (or BufferEB) was added, allowed to stand for 1 minute, and centrifuged at 13,000 rpm for 30 to 60 seconds to collect a second strand cDNA solution. The recovered solution was transferred to a 1.5 ml PCR tube and allowed to stand on ice.

(4) Transcriptional synthesis of aRNA For transcriptional synthesis, Ambion's MEGAscript T7 Kit (# 1334, manufactured by Ambion) was used.

The reagents shown in Table 4 were added to a 0.2 ml-PCR tube and incubated at 37 ° C. for 6 hours to overnight.

（５）aRNAの精製
aRNAの精製には、RNeasy QIAGEN MiniElute^TM Purification Kit (QIAGEN社製 #74204)を使用した。（４）で得られたaRNAに80μｌのDEPC処理水を加えて、100μｌにメスアップした。滅菌済みの空の1.5mlチューブにこの溶液をすべて移し入れ、350μｌのBuffer RLTを加えて、よく混合した。さらに250μｌの99.5%エタノールを加えて、ピペッティングにより混合した。RNeasy MiniElute^TM スピンカラムを2mlのcollection tubeに差し込み、上記の混合液を入れ、15秒間、10,000rpmで遠心した。溶出液を捨て、再度そのRNeasy MiniElute^TMスピンカラムを新しいcollection tubeに差し込み、500μｌのBuffer RPEを加え、15秒間、10,000rpmで遠心した。溶出液を捨て、500μｌの80%エタノールを加え、2分間、10,000rpmで遠心した。フィルター上の余分なエタノールを完全に除くため、５分間、13,000rpmで遠心した。新しい1.5mlチューブにそのRNeasy MiniElute^TM スピンカラムを差し込み、14μlのDEPC処理水を加え、1分間放置後、3分間、13,000rpmで遠心して、液を回収した。次に分光光度計で濃度を測定し、3.2μg/μlのaRNAを得た。 [Table 4]

(5) Purification of aRNA
For purification of aRNA, RNeasy QIAGEN MiniElute ^™ Purification Kit (QIAGEN # 74204) was used. 80 μl of DEPC-treated water was added to the aRNA obtained in (4) to make up to 100 μl. All of this solution was transferred to a sterilized empty 1.5 ml tube and 350 μl Buffer RLT was added and mixed well. Further 250 μl of 99.5% ethanol was added and mixed by pipetting. An RNeasy MiniElute ^™ spin column was inserted into a 2 ml collection tube, the above mixture was added, and centrifuged at 10,000 rpm for 15 seconds. The eluate was discarded, and the RNeasy MiniElute ^™ spin column was again inserted into a new collection tube, 500 μl of Buffer RPE was added, and the mixture was centrifuged at 10,000 rpm for 15 seconds. The eluate was discarded, 500 μl of 80% ethanol was added, and the mixture was centrifuged at 10,000 rpm for 2 minutes. In order to completely remove excess ethanol on the filter, it was centrifuged at 13,000 rpm for 5 minutes. The RNeasy MiniElute ^™ spin column was inserted into a new 1.5 ml tube, 14 μl of DEPC-treated water was added, and the mixture was allowed to stand for 1 minute and then centrifuged at 13,000 rpm for 3 minutes to collect the liquid. Next, the concentration was measured with a spectrophotometer to obtain 3.2 μg / μl of aRNA.

3. Production of Hollow Fiber Bundle A hollow fiber bundle was produced using the array fixture shown in FIG. In the figure, x, y, and z are orthogonal three-dimensional axes, and the x-axis coincides with the longitudinal direction of the fiber.

  First, two perforated plates (50) each having a thickness of 0.32 mm and a total of 144 holes in 12 rows each in a vertical and horizontal direction with a hole (52) having a center distance of 0.42 mm are prepared. did. These perforated plates were overlapped, and polycarbonate hollow fibers (54) (added by 1% by mass of carbon black manufactured by Mitsubishi Engineering Plastics) were passed through each of the holes one by one.

  The position of the two perforated plates was moved in a state in which a tension of 0.1 N was applied to each fiber in the X-axis direction, and was fixed at two positions of 20 mm and 100 mm from one end of the hollow fiber. . That is, the interval between the two perforated plates was 80 mm.

  Next, the three surrounding surfaces of the space between the perforated plates were surrounded by a plate-like object (56). In this way, a container having an open top only was obtained.

  Next, the resin raw material was poured into the container from the upper part of the container. As resin, what added 2.5 mass% carbon black was used with respect to the total weight of polyurethane resin adhesives (Nippon Polyurethane Industry Co., Ltd., Nippon-Poran 4276, Coronate 4403). The resin was cured by standing at 25 ° C. for 1 week. Next, the porous plate and the plate-like material were removed to obtain a hollow fiber bundle. One of the fiber ends of this hollow fiber bundle was sealed and the other was open.

4). Preparation of Gel Precursor Solution Containing Probe and Polymer Compound A gel precursor solution having the composition shown in Table 5 was prepared. In Table 5, Reference Examples 1 and 2 were set as standards for confirming the removal of aRNA described later.

５．ゲル充填中空繊維束の製造
４．にて調製したゲル前駆体溶液を、３８４マイクロタイタープレートの所定位置のウェルに図２に示した通りに、各条件につき３ウェルに８０μｌ分注した。 [Table 5]

5. 3. Production of gel-filled hollow fiber bundles As shown in FIG. 2, 80 μl of the gel precursor solution prepared in 3 was dispensed into wells at predetermined positions of a 384 microtiter plate as shown in FIG.

  Next, the fiber end of the hollow fiber bundle that was opened was fixed in a state of being immersed in the gel precursor solution, and placed in a desiccator. Next, after the desiccator was depressurized, nitrogen gas was sealed in the desiccator, the system was returned to normal pressure, and the gel-forming solution was introduced into the hollow fiber hollow part. Subsequently, the inside of a container was made into 70 degreeC and the polymerization reaction was performed for 3 hours. Thus, a gel-filled hollow fiber bundle in which the probe was immobilized on the gel was obtained.

6). Thinning 5. The hollow fiber bundle obtained in (1) was sliced in a direction perpendicular to the longitudinal direction of the fiber using a microtome to obtain 50 microarrays having a thickness of 0.5 mm.

7). 5. Removal of aRNA from gel Next, according to the following steps a) to d), 6. The aRNA removal treatment was performed in the gel of the thin piece obtained in (1).

a) Mix 500 μl of Ambion 10 × RNA Fragment reagent and 9.5 ml of sterilized water in a petri dish and heat to 70 ° C.

b) Immerse 6 chips in this solution and incubate at 70 ° C.

c) The chip is taken out every 60 minutes, and the taken-out chip is immersed in 2 × SSC for 1 hour.

d) Further, the above operations b) and c) are repeated three times, and the treatment is performed for a total of 180 minutes.

In order to confirm whether aRNA was removed, a solution was prepared by dissolving SYBR Gold at a 10,000-fold dilution in 8 ml of 2 × SSC solution, and the treated chip was stained. After immersion for 30 minutes in 2 × SSC, with a fluorescent microscope equipped with a CCD camera, using a filter of Cy ^TM 3, it was measured to detect fluorescence intensity at an exposure time 10 msec. The measurement results are shown in FIG. After 120 minutes of treatment, the fluorescence intensity of Reference Example 1 was equivalent to the fluorescence intensity of Reference Example 2, and it was determined that aRNA was almost removed. Therefore, the aRNA removal treatment should be performed for 120 minutes. The microarray obtained in 1 was processed.

8). Sample Preparation Next, a sample to be subjected to hybridization was prepared.

(1) Synthesis of First Strand cDNA from Total RNA In this experiment, from 2 μg of total RNA derived from yeast (manufactured by Clontech), the above 2. First strand cDNA was synthesized by the same operation as in (1).

(2) PCR amplification of cDNA Next, PCR amplification was performed using the first strand cDNA as a template and the following primers (SEQ ID NOs: 2 and 3).

Primer 1 (SEQ ID NO: 2); TAATACGACT CACTATAGGG TTCTATCAAC CCGGATGAGG
Primer 2 (SEQ ID NO: 3); CCAGCACCGTA AAATTCGT
PCR was performed under the following conditions.

（条件）［94℃：10min］→［94℃：30秒］→［62℃：30秒（30サイクル）］→［72℃：30秒］→［72℃：5min］→［4℃：∞］。ＰＣＲ反応終了後、QIAGEN PCR purification Kitで精製 50μlのcＤＮＡ溶液を得た。 [Table 6]

(Conditions) [94 ° C: 10 min] → [94 ° C: 30 sec] → [62 ° C: 30 sec (30 cycles)] → [72 ° C: 30 sec] → [72 ° C: 5 min] → [4 ° C: ∞ ]. After completion of the PCR reaction, a 50 μl purified cDNA solution was obtained using the QIAGEN PCR purification Kit.

(3) aRNA transcription synthesis
Using the Ambion MEGAscript T7 Kit (Ambion # 1334), the reagents in Table 7 were added to a 0.2 ml-PCR tube and incubated at 37 ° C. for 6 hours to overnight.

（４）aＲＮＡの精製
２．（５）と同様の操作により、aＲＮＡを精製して、検体aＲＮＡを得た。 [Table 7]

(4) Purification of aRNA The aRNA was purified by the same operation as (5) to obtain a sample aRNA.

9. Hybridization using aRNA sample (1) Fragmentation of aRNA sample
Using Ambion RNA Fragmentation Reagents (Ambion # 8740), fragmentation was performed under the conditions shown in Table 8. After incubation at 70 ° C. for 5 minutes, 2 μl of Stop Solution attached to the kit was added.

（２）検体溶液の調整とハイブリダイゼーション操作
（１）で調製した断片化aRNA溶液に、表９に記載の各溶液を混合し、100μlとした。 [Table 8]

(2) Preparation of Sample Solution and Hybridization Operation Each solution shown in Table 9 was mixed with the fragmented aRNA solution prepared in (1) to make 100 μl.

The final concentration of SSC and SDS in the solution is 2 × SSC, 0.2% SDS.

この溶液を94℃で2分間加熱した後、37℃まで冷却した。次いで、７．で処理したマイクロアレイと共にハイブリパックに封入し、65℃の遮光されたインキュベーター内で17時間、静置し、ハイブリダイゼーションを行った。 [Table 9]

The solution was heated at 94 ° C. for 2 minutes and then cooled to 37 ° C. Then, 7. It was enclosed in a hybrid pack together with the microarray treated in step 1, and left to stand in a light-shielded incubator at 65 ° C. for 17 hours for hybridization.

(3) Washing operation The chip was taken out from the hybrid pack, immersed in a cylindrical case containing 15 ml of 2 × SSC / 0.2% SDS solution that had been brought to 45 ° C., kept warm at 45 ° C., and allowed to stand for 20 minutes. Next, the tube was replaced with a new cylindrical case containing 15 ml of 2 × SSC / 0.2% SDS solution that had been kept at 45 ° C., and further washed at 45 ° C. for 20 minutes. Finally, it was replaced with a cylindrical case containing 15 ml of 2 × SSC solution that had been kept at 45 ° C., and washed at 45 ° C. for 10 minutes.

10. Detection Operation After the hybridization treatment, nucleic acid hybrids were detected using a fluorescence detection apparatus.

(1) Microarray staining treatment Before detection with a detection device, staining was performed using a streptavidin-Cy ^TM 5 labeling reagent manufactured by Amersham. The microarray was immersed in Streptavidin-Cy ^™ 5 labeling reagent buffer and stained at room temperature for 30 minutes. After the staining, the immersion operation for 5 minutes was performed 4 times in the washing buffer shown in Table 10 at room temperature.

（２）蛍光検出
（１）で蛍光標識されたマイクロアレイを三菱レイヨン社製冷却ＣＣＤ式蛍光検出装置で各スポットの蛍光強度を測定した。 [Table 10]

(2) Fluorescence detection The fluorescence intensity of each spot of the microarray fluorescently labeled in (1) was measured with a cooled CCD fluorescence detector manufactured by Mitsubishi Rayon Co., Ltd.

<Detection conditions>
・ Fluorescent filter; Cy ^TM 5 filter ・ Exposure time: 340msec
(3) Detection results The fluorescence detection results are shown in FIG. In the figure, “B” indicates a blank spot result. In the figure, the fluorescence intensity is shown as an average value of three spots. From the result of FIG. 4, the gel (Examples 1 and 2) provided with a space with aRNA is about 2 to 3.5 times higher in fluorescence intensity than the gel with no space provided with aRNA (Comparative Example 1). The result was high. Further, from the results of Examples 1 and 2, it was confirmed that the fluorescence intensity increased in proportion to the addition ratio of aRNA. This result suggests that the spatial freedom in the gel matrix was improved by adding aRNA during gelation.

It is the perspective view which showed the arrangement | sequence fixing tool. It is the figure which showed the fixed position of the probe. It is the figure which showed the removal process result of aRNA. It is the figure which showed the fluorescence detection result of the microarray.

Explanation of symbols

50 ... perforated plate 52 ... hole 54 ... hollow fiber 56 ... plate-like material

SEQ ID NO: 1 synthetic DNA
SEQ ID NO: 2 Synthetic DNA
SEQ ID NO: 3 Synthetic DNA

Claims (5)

  A probe-immobilized gel obtained by preparing a gel containing a probe and a polymer compound and then removing the polymer compound from the gel.   The gel according to claim 1, wherein the polymer compound has binding properties to the probe.   The gel holding | maintenance tubular body with which the gel of Claim 1 or 2 is hold | maintained at the tubular body.   A gel microarray in which a plurality of the gels according to claim 1 or 2 are fixed to a substrate.   A gel microarray in which the gel according to claim 1 or 2 is held in a through hole of a substrate having a plurality of through holes.

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