JP2001161361A - Method for hybridization of biopolymer chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for hybridization of a material containing a probes. SOLUTION: This method for hybridization of a biopolymer carrier (chip) obtained by binding biopolymer probes on a carrier is a method for detecting biopolymer chip characterized by transferring specimens into the chip by a method other than natural diffusion and fleetly removing the specimens forming no hybrid with the biopolymer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for hybridizing a probe-containing substance. Specifically, in a hybridization method of a biopolymer support having a biopolymer probe bound to a carrier (hereinafter referred to as a biopolymer chip), a sample is diffused into the biopolymer chip by a method other than natural diffusion. The present invention relates to a method for detecting a biopolymer chip, which comprises forming a hybrid and immediately removing a specimen not bound to the biopolymer probe to the outside of the chip system.

[0002]

2. Description of the Related Art In recent years, genome projects for various organisms have been promoted, and a large number of genes including human genes and their base sequences have been rapidly identified. The function of a gene whose sequence has been clarified can be examined by various methods. As one of the promising methods, gene expression analysis using the clarified nucleotide sequence information is known. For example, methods utilizing various nucleic acid-nucleic acid hybridization reactions and various PCR reactions, as represented by Northern hybridization, have been developed, and the method is used to examine the relationship between various genes and their biological function expression. Can be. However, these methods have limitations on the number of genes that can be applied. Therefore, in view of the entire complex reaction system consisting of a large number of genes at the individual level, which is being revealed through today's genome project, comprehensive and systematic analysis of genes by the above method is necessary. It is difficult.

[0003] Recently, a new analysis method or methodology called a DNA microarray method (DNA chip method) that enables simultaneous expression analysis of a large number of genes has been developed and attracted attention. These methods are, in principle, the same as the conventional methods in that they are nucleic acid detection / quantification methods based on nucleic acid-nucleic acid hybridization reaction, but a large number of microarrays or chips are mounted on a flat base plate. There is a great feature in that a DNA fragment in which DNA fragments are aligned and fixed at high density is used. As a specific method of using the microarray method, for example, a sample in which the expressed gene or the like of a cell to be studied is labeled with a fluorescent dye or the like is hybridized on a flat substrate, and mutually complementary nucleic acids (DNA or RNA) are bound to each other. A method of labeling the portion with a fluorescent dye or the like, and then reading the portion at high speed with a high-resolution analyzer. Thus, the amount of each gene in the sample can be quickly estimated. With the introduction of this new method, it has become possible to reduce the amount of the reaction sample and to analyze and quantify the reaction sample in a large amount, quickly and systematically with good reproducibility.

[0004] Some of the present inventors have previously developed and applied for a novel microarray manufacturing method. (Japanese Patent Application No. 11-
No. 84100) The present invention is to produce a nucleic acid-immobilized gel holding hollow fiber array holding a nucleic acid-immobilized gel therein, and to obtain a slice by cutting the array in a direction intersecting the fiber axis of the array. . This slice is an immobilized nucleic acid two-dimensional high-density array, ie, a microarray. On the other hand, an attempt has been made to immobilize a probe nucleic acid in a gel and detect hybridization with a nucleic acid in a sample. (JP-A-3-47097, WO98 / 51823)

[0005]

However, when performing a gene analysis using a currently used microarray, it takes a long time for the hybridization and the washing operation after the hybridization.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a nucleic acid-immobilized gel is held in a porous hollow fiber proposed by some of the present inventors. Nucleic acid-immobilized gel holding hollow fiber array (Japanese Patent Application No. Hei 11
-84100) That is, in the DNA chip, hybridization is performed in a short time by forcibly moving the sample DNA to the vicinity of the probe DNA,
Furthermore, they found that the sample DNA in which no hybridization occurred could be quickly removed from the chip, and completed the present invention.

That is, the present invention relates to a method for hybridization of a probe-containing substance, particularly a biopolymer chip, in which a specimen is transferred into the chip by a method other than spontaneous diffusion to form a hybrid, and the probe is hybridized with the biopolymer chip. The present invention relates to a method for hybridization of a biopolymer chip, wherein a specimen not formed is promptly removed from the chip.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. The type and form of the probe-containing substance in the present invention are not particularly limited as long as the method of the present invention is applied. The biopolymer probe in the present invention includes, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RN)
A), peptide nucleic acids, proteins (enzymes, antibodies, etc.), antigens, polysaccharides and the like. When applying a voltage to both surfaces of the biopolymer chip when performing hybridization between the sample and the probe, for example, a submarine-type electrophoresis tank (FIG. 1) or a blotting apparatus (FIG. 2) can be used. is there. The device is not limited to these. The specimen is adsorbed on a filter paper or a membrane, or entrapped in a high molecular polymer such as acrylamide or agarose, and adhered to a biopolymer chip. Next, after applying a voltage for a certain period of time, the hybridization location is specified by a detector. Further, by disposing a water-absorbing substance on one surface of the biopolymer chip, a sponge, paper, or the like can be used as the water-absorbing substance when a sample placed on the opposite side is moved into the chip to perform a binding reaction.

[0009]

The present invention will now be described in more detail with reference to the following examples.
explain. However, the invention is limited to only these examples.
It is not something to be done. Example 1 (1) Preparation of chromosomal DNA Rhodococcus rhodochrous J-1 (FERM BP
-1478) in a nutrient medium (glucose 15 g, yeast
1g, sodium glutamate 10g, KH ₂PO₄
 0.5g, K₂HPO₄ 0.5g, MgSO₄・ 7
H₂O 0.5 g / L pH 7.2)
The cells were cultured at 0 ° C. for 3 days and collected. Chromosomal DNA from the cells
Was prepared and used as a template for PCR.

(2) Preparation of Probe FIG. 3 shows the positions of oligonucleotides synthesized for probe preparation. Oligonucleotide A (SEQ ID NO: 1) is about 400 times less than oligonucleotide B (SEQ ID NO: 2).
Located upstream of the base, oligonucleotide E (SEQ ID NO: 3) is 400 bases from oligonucleotide A,
Oligonucleotide F (SEQ ID NO: 4) is located 600 bases downstream from oligonucleotide B. PCR
The oligonucleotides A and B were oligonucleotide oligonucleotides whose 5 ′ ends were modified with acrylamide (consigned synthesis to Wako Pure Chemical Industries). PCR was performed using Asymmetric PC in which one primer was present in excess.
R was performed. The primer concentration was 5 ′ acrylamide modified oligonucleotide A-oligonucleotide E (10
0: 1) or 5 ′ acrylamide modified oligonucleotide B-oligonucleotide F (100: 1). Other conditions were in accordance with the specifications of Ex-Taq (Takara Shuzo Co., Ltd.) and TaKaRa PCR Thermal.
Performed using a CyclerPERSONAL. The reaction was performed in 100 μl, and the temperature condition was 93 ° C. for 30 seconds, 65 ° C.
40 cycles of 30 seconds at 72 ° C and 2 minutes at 72 ° C were performed. By this PCR, 5 ′ of about 400 bases (probe G: SEQ ID NO: 5) and 600 bases (probe H: SEQ ID NO: 6)
Acrylamide modified probe DNA was amplified.

(3) Preparation of nucleic acid-immobilized flake Aqueous solution A (acrylamide 3.7) having the following composition
Parts by mass, methylene bisacrylamide 0.3 parts by mass,
0.1 parts by mass of 2,2′-azobis (2-amidinopropane) dihydrochloride, probe G or probe H 0.00
5 parts by mass). Polyethylene porous hollow fiber membrane MHF200T having non-porous intermediate layer in aqueous solution A
L (Mitsubishi Rayon Co., Ltd., outer diameter 290 μm, inner diameter 2
(00 μm), and then transferred to a sealed glass container whose inside was saturated with water vapor, and left at 80 ° C. for 4 hours to carry out a polymerization reaction.

The gel in which the probe G or H was immobilized was retained in the porous layer on the inner side of the obtained porous hollow fiber membrane as compared with the hollow portion and the non-porous intermediate layer. Porous hollow fiber 5 holding gel immobilized with probe G obtained by the method
The books were arranged in close contact with each other without overlapping on the CFC plate, and both ends were fixed. A polyurethane resin adhesive (Coronate 4403, Nippon Polyurethane Industry Co., Ltd., Nipporan 4223) was thinly applied thereto, and the nucleic acid-immobilized porous hollow fibers were bonded. After the polyurethane resin has hardened sufficiently,
The sheet was peeled off from the Teflon plate to obtain a sheet in which porous hollow fibers holding the nucleic acid-immobilized gel were arranged in a line. Similarly, for the probe H, a sheet was obtained in which the porous hollow fibers holding the nucleic acid-immobilized gel were arranged in a line. Also,
As a blank, a similar sheet without immobilized nucleic acid was prepared.

Next, these sheet-like materials are blanked,
Probe G, blank, probe H, blank in the order of 5
The porous hollow fiber array body holding a nucleic acid-immobilized gel in which a total of 25 nucleic acid-immobilized porous fibers are regularly arranged in a square in a five-by-five matrix is obtained by adhering with the above adhesive. Was. The porous hollow fiber array holding the nucleic acid-immobilized gel thus obtained was cut out to a thickness of 0.1 mm using a microtome, and the porous hollow fiber array holding a total of 400 nucleic acid-immobilized gels, 20 in each of length and width, was cut out. A thin section in which the cross sections of the hollow fibers were regularly arranged was obtained.

(4) Preparation of Fluorescently Labeled Specimen The following specimen DNA was prepared as a model of the specimen nucleic acid and used for hybridization. To prepare a sample that hybridizes only to probe G, PCR is performed using oligonucleotide E and oligonucleotide A,
Sample I (SEQ ID NO: 7) of about 400 bases was prepared. For preparation of a sample that hybridizes only to probe H, oligonucleotide F and oligonucleotide B were used to prepare a PC.
R was performed to prepare a sample J (SEQ ID NO: 8) of about 600 bases.

In order to fluorescently label a specimen, oligonucleotides E and F whose 5 'ends are labeled with Cy5 (Cy5-oligonucleotide E, Cy5
-Oligonucleotide F) was synthesized (Amersham Pharmacia Biotech, OlidoExpress), and P
Used for CR. PCR was performed using primer concentration Cy5-
Oligonucleotide E-oligonucleotide A (100: 1) or Cy5-oligonucleotide F-oligonucleotide B (100: 1) was prepared, and the others were prepared according to the specifications of Ex-Taq (Takara Shuzo Co., Ltd.). PCR Thermal
Performed using Cycler PERSONAL. The reaction was performed in 100 μl, and the temperature condition was 93 ° C. for 30 seconds, 6
40 cycles of 5 ° C. for 30 seconds and 72 ° C. for 2 minutes were performed. The DNA of sample I and sample J was amplified by this PCR. The reaction-terminated liquid was removed from GFX PCR DNA a using SUPEC-02 (Takara Shuzo) to remove unreacted primers.
Second Gel Band Purification
Collected by Kit (Amersham Pharmacia Biotech).

(5) Hybridization 2 μl of the collected fluorescently labeled sample was filtered with filter paper (PastTra).
nsfer Filter Paper: Amersham Pharmacia Biotech) and Example 1 (3)
The DNA-immobilized lamella obtained in the above was brought into close contact with one side, and a voltage of 5 V was applied for 2 hours using the apparatus shown in FIG. 1 to perform hybridization and washing, followed by observation with a fluorescence detector (fluorescence microscope). .

As a result, the probe G in the nucleic acid-immobilized slice was
It was confirmed that sample I was specifically hybridized only to the cross section of the porous fiber where probe was immobilized, and sample J was hybridized specifically only to the cross section of the porous fiber where probe H was immobilized.

[0018]

According to the present invention, in the probe-containing hybridization method, the hybridization is performed in a short time by forcibly moving the sample to the vicinity of the probe, and furthermore, no hybridization occurs. The specimen can be quickly removed.

[0019]

[Sequence List] SEQUENCE LISTING <110> Mitsubishi Rayon Co., Ltd. <120> Hybridization method of biopolymer <130> P110682000 <210> 1 <211> 33 <212> DNA <213> Rhodococcus rhodochrous <220> <223> Synthetic DNA <400> 1 gcgatcgaaa ccttgctgta cgagcgaggg ctc 33 <210> 2 <211> 32 <212> DNA <220> <213> Rhodococcus rhodochrous <223> Synthetic DNA <400> 2 gatgaggtgg aggtcagggt ttgggacagc <ag> 32 <210> 211> 31 <212> DNA <213> Rhodococcus rhodochrous <220> <223> Synthetic DNA <400> 3 gctcaagcgc gatttcggtt tcgacatccc c 31 <210> 4 <211> 30 <212> DNA <213> Rhodococcus rhodochrous <220> < 223> Synthetic DNA <400> 4 catgtcgcgt cgttgttgga cgaagcggta 30 <210> 5 <211> 388 <212> DNA <213> Artificaial Sequence <220> <223> Synthetic DNA <400> 5 gcgatcgaaa ccttgctgta ccttgctgta cgagcgaggg ctcatcacgc gtcat gc gc gc ctcatcaggc ctcatcacggc cccat gcgc ctcatcacgg cc gatcggcccg atgggcggtg ccaaggtcgt ggccaagtcc 120 tgggtggacc ctgagtaccg caagtggctc gaagaggacg cgacggccgc gatggcgtca 180 ttgggctatg ccggtgagca ggcacaccaa atttcggcgg tcttcaacga aacg 240 catcacgtgg tggtgtgcac tctgtgttcg tgctatccgt ggccggtgct tggtctcccg 300 cccgcctggt acaagagcat ggagtaccgg tcccgagtgg tagcggaccc tcgtggagtg 360 ctcaagcgc11 ag <attr> aggtcagggt ttgggacagc agctccgaaa tccgctacat cgtcatcccg 60 gaacggccgg ccggcaccga cggttggtcc gaggaggagc tgacgaagct ggtgagccgg 120 gactcgatga tcggtgtcag taatgcgctc acaccgcagg aagtgatcgt atgagtgaag 180 acacactcac tgatcggctc ccggcgactg ggaccgccgc accgccccgc gacaatggcg 240 agcttgtatt caccgagcct tgggaagcaa cggcattcgg ggtcgccatc gcgctttcgg 300 atcagaagtc gtacgaatgg gagttcttcc gacagcgtct cattcactcc atcgctgagg 360 ccaacggttg cgaggcatac tacgagagct ggacaaaggc gctcgaggcc agcgtggtcg 420 actcggggct gatcagcgaa gatgagatcc gcgagcgcat ggaatcgatg gccatcatcg 480 actgacatcc cctgtgtctc catctagcag cagtgcgggc gtaccccgac ggtgctgagc 540 cgacggggta cgcccgcact tcatcaatga cggtggttcc taatttggc cgtg gtg gtg gtg cgtg gtg gtg gtg 1> 388 <212> DNA <213> Artificaial Sequence <220> <223> Synthetic DNA <400> 7 ggatgtcgaa accgaaatcg cgcttgagca ctccacgagg gtccgctacc actcgggacc 60 ggtactccat gctcttgtac caggcgggcg ggagaccaag caccggccac ggatagcacg 120 aacacagagt gcacaccacc acgtgatgcg tttgggagtc gttgaagacc gccgaaattt 180 ggtgtgcctg ctcaccggca tagcccaatg acgccatcgc ggccgtcgcg tcctcttcga 240 gccacttgcg gtactcaggg tccacccagg acttggccac gaccttggca ccgcccatcg 300 ggccgatctc gttctcgtag tacgaaacga ctcggtcgac cgcggcgggc gtgatgagcc 360 ctcgctcgta cagcaaggtt tcgatcgc 388 <210> 8 <211> 611 <212> DNA <213> Artificaial Sequence <220> <223> Synthetic DNA <400> 8 ccgcgagatc agtatccacc gagccaaatt aggaaccacc gtcattgatg aagtgcgggc 60 gtaccccgtc ggctcagcac cgtcggggta cgcccgcact gctgctagat ggagacacag 120 gggatgtcag tcgatgatgg ccatcgattc catgcgctcg cggatctcat cttcgctgat 180 cagccccgag tcgaccacgc tggcctcgag cgcctttgtc cagctctcgt agtatgcctc 240 gcaaccgttg gcctcagcga tggagtgaat gagacgctgt cggaagaact cccattcgta 300 cgacttctga tccgaaagcg cgatggcg ac cccgaatgcc gttgcttccc aaggctcggt 360 gaatacaagc tcgccattgt cgcggggcgg tgcggcggtc ccagtcgccg ggagccgatc 420 agtgagtgtg tcttcactca tacgatcact tcctgcggtg tgagcgcatt actgacaccg 480 atcatcgagt cccggctcac cagcttcgtc agctcctcct cggaccaacc gtcggtgccg 540 gccggccgtt ccgggatgac gatgtagcgg atttcggagc tgctgtccca aaccctgacc 600 tccacctcat c 611

[0020]

[Free text of Sequence Listing] SEQ ID NO: 1: Synthetic DNA SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3: Synthetic DNA SEQ ID NO: 4: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 7: Synthetic DNA sequence No. 8: synthetic DNA

[Brief description of the drawings]

FIG. 1 shows a submarine type electrophoresis tank.

FIG. 2 shows a blotting device.

FIG. 3 shows a method for producing a probe.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) (C12N 1/20 C12N 15/00 A C12R 1:01) G01N 27/26 325E F-term (reference) 4B024 AA11 AA20 BA80 CA03 DA05 GA19 HA14 HA19 4B029 AA07 AA23 BB20 CC05 CC12 FA15 4B063 QA01 QA08 QA18 QQ42 QQ60 QR32 QR39 QR56 QR62 QR85 QS03 QS15 QS16 QS25 QS34 QS36 QS39 QX02 4B045 AA45XA45A

Claims (8)

[Claims] 1. A method for hybridization of a substance containing a probe, wherein the specimen hybridizes with the probe by diffusing the substance in the substance by a method other than spontaneous diffusion, and the specimen unbound to the probe contains the specimen. A method for hybridization of a substance containing a probe, wherein the substance is removed from the system. 2. The method for hybridizing a substance containing a probe according to claim 1, wherein the substance containing a probe is a substance obtained by binding a biopolymer probe to a carrier. 3. The carrier according to claim 2, wherein the carrier is a water-soluble polymer gel.
A hybridization method for a substance containing the probe as described above. 4. The method for hybridizing a substance containing a probe according to claim 3, wherein the water-soluble polymer is acrylamide. 5. A method for hybridizing a substance containing a probe according to claim 2, wherein the biopolymer is a nucleic acid. 6. A method for hybridizing a substance containing a probe according to claim 1, wherein the specimen is fluorescently labeled. 7. The method for hybridization of a substance containing a probe according to claim 1, wherein the method other than the natural diffusion is by voltage. 8. The method for hybridization of a substance containing a probe according to claim 1, wherein the transfer method other than the natural diffusion is by a water-absorbing substance.