WO2002046753A1

WO2002046753A1 - Functional dna array

Info

Publication number: WO2002046753A1
Application number: PCT/JP2001/010600
Authority: WO
Inventors: Junichi Mineno; Masatomo Rokushima; Shusaku Yamashita; Tatsuji Enoki; Hiroaki Sagawa; Jean-Jacques Farhi; Ikunoshin Kato
Original assignee: Takara Bio Inc.
Priority date: 2000-12-06
Filing date: 2001-12-05
Publication date: 2002-06-13
Also published as: AU2002221046A1

Abstract

A DNA array wherein one or more genes or fragments thereof, which are selected due because of showing a difference in base sequence or expression dose between the subjects to be compared with each other, have been immobilized in a preliminarily determined region on a support.

Description

Description Functionality DNA Array Technical Field

The present invention relates to an array of DNAs sorted by function, useful for analysis of gene functions such as genome analysis and disease state analysis, a method for producing the array, and an array development system. Background art

Due to the development of genetic engineering, the technological development of genetic information analysis and genetic information management systems has been rapidly progressing in recent years. The accumulated genetic information is, for example, trying to make a significant difference in drug development. In addition, genes related to diseases are being found one after another, and attempts are being made to identify promising therapeutic genes and their products, and to efficiently find targets for drug development. Combining targets found from genetic information with rapidly expanding new drugs, such as antibodies, antisense nucleic acids, receptor technologies, and gene therapy, breakthroughs that could not be achieved with existing drugs Can be expected to lead to new products. In addition, it is possible to classify diseases at the genetic level and to administer therapeutic drugs that match them.

In recent years, DNA arrays have been developed as a means for analyzing gene information, and many high-density DNA arrays are commercially available.

These DNA arrays can be classified as follows depending on the method of selecting the DNA to be fixed.

The first is a DNA array that covers the entire genome or open reading frame of a species.

This DNA array has the advantage of being able to analyze the expression of all genes of the target species, but requires DNA sequence information of the entire genome, and has a high manufacturing cost due to the large number of DNA types to be immobilized. There are drawbacks such as high. Species that can produce such a DNA array are virtually prokaryotic at best, eukaryotic microorganisms. It is limited to small genome size.

The second is to immobilize DNA randomly selected from cDNA libraries and genomic DNA libraries.

This DNA array has the advantages that it does not require the sequence information of the DNA to be immobilized and that the number of DNAs to be immobilized can be freely set.

For example, when randomly selected DNA is immobilized, the difference in gene expression between the two samples is used for analysis. There are many. Also, since it does not cover all genes, no information on selectivity or missing genes can be obtained. Furthermore, particularly when selecting DNA to be immobilized from a cDNA library, cDNA derived from mRNA with a high expression level is spotted repeatedly, and only a small amount of information is obtained for the number of spots on the DNA array. There is no danger. On the other hand, the risk of repeatedly spotting highly expressed genes can be reduced by selecting the DNA to be immobilized from a uniform (normalization) cDNA library. It is even more reliable if the DNA sequence is determined and the DNA having the same sequence is eliminated in advance and then fixed. However, even with these improvements, the other drawbacks of the second DNA array remain problematic.

At present, huge amounts of money are required for gene information, such as genomic analysis, and even the most well-funded pharmaceutical and chemical companies are burdened with large costs.

On the other hand, the 21st century is the age of biotechnology and genes, and genes are also a common property of mankind. There is a need for a method of obtaining genomic analysis information that reduces risks and is not limited to large-capacity pharmaceutical and chemical companies, but is open to various R & D companies.

Currently, commercially available DNA arrays are mainly arrays in which DNAs whose DNA sequences are determined in advance are immobilized in predetermined regions, so-called plain arrays. At present, no array has been developed or provided in which the specified DNA is immobilized in a predetermined area.

Therefore, there is a need to provide a system that sorts genes by function (sort) and develops an array in which the sorted genes are immobilized inexpensively and easily. In addition, the development of DNA arrays takes a long time to select the DNA to be used, and in the case of producing high-density DNA microarrays, the production of DNA to be immobilized on an array requires a great deal of labor and cost. It is.

Therefore, many pharmaceutical and chemical companies that are trying to expand their business using gene function analysis are still struggling to obtain the desired DNA arrays. Purpose of the invention

In view of the above problems, an object of the present invention is to provide a DNA array in which genes sorted according to functions that enable diversification, efficiency, and risk reduction in genome analysis are fixed, that is, functions. An object of the present invention is to provide a development system for a sex DNA array. Summary of the Invention

An overview of the present invention is that, in the first invention of the present invention, one or more genes or fragments thereof selected as having a difference in the nucleotide sequence or expression level between the comparison subjects are previously determined on a carrier. It relates to a DNA array immobilized in a region.

In the first invention of the present invention, those in which the comparison object is two or more types of cells or tissues can be suitably used. Further, the comparison object may be two or more cells or tissues having different origins. In addition, at least one of the comparison targets may be a cell or a tissue derived from a lesion site. Furthermore, at least one of the comparison targets may be an artificially treated cell or tissue.

A second invention of the present invention is a method for producing a functional DNA array according to the first invention of the present invention,

(1) a step of selecting a gene having a different base sequence or its expression level between comparison targets; (2) a step of preparing the gene selected in (1) above or a fragment thereof; and

(3) a step of immobilizing the gene or the fragment thereof prepared in (2) above on a predetermined region on a carrier.

A third invention of the present invention is a functional DNA array development system,

The consortium founded by the founders (consortiurn) is a consortium Recruiting Caro members and raising funding from consortium members, the consortium orders a functional DNA array developer to produce a functional D NAT ray and, if necessary, provides information and / or Providing a sample containing a test gene to a functional D NAT ray developer,

The functional DNA array developer sorts the test genes by function to produce a functional DNA array, manufactures a functional DNA array using the genes sorted by the function, and provides exclusive access to the consortium. provide,

The present invention relates to a functional D NAT ray development system. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a functional DNA array development system of the present invention.

FIG. 2 is a diagram showing a flowchart of the development system of the present invention. Detailed description of the invention

In the present invention, a functional D NAT ray refers to a gene that has a difference in base sequence or expression level between comparison targets, selects the gene, and fixes the gene or a fragment thereof in a predetermined region on a carrier. Refers to an array that has been converted As described above, in the DNA array of the present invention, the genes selected as having a difference in the nucleotide sequence or expression level between the comparison targets are immobilized, so that the difference in the nucleotide sequence or expression level of the gene is obtained. This is different from the conventional DNA array that was made without considering the above.

Although not particularly limited, for example, a functional DNA array for analyzing single nucleotide substitution polymorphisms related to disease morbidity, drug susceptibility, side effects on drugs, and a functional group for analyzing gene expression regulatory units DNA arrays, functional DNA arrays for disease classification analysis, functional DNA arrays for disease classification, functional DNA arrays for analyzing differences between different cell types, cells in specific biological environments Functional DNA array for analyzing the state of the disease, functional DNA array for analyzing the response of cells to a specific stimulus, functional DNA array for analyzing the pathological condition, and gene expression over time Functional DNA arrays and the like for analyzing changes are exemplified. According to the present invention, a test DNA is sorted by function, and a functional DNA array using the genes sorted by the function is provided. The functional DNA array is exemplified by the functional DNA array described above.

The functional DNA array provided by the present invention is characterized in that, for genes sorted by function, their base sequences are used irrespective of their known ability or not. In the present invention, sorting by function refers to classifying genes having a difference in base sequence or expression level between comparison targets.

As used herein, the term “DNA array” refers to an array in which genes or DNA-derived DNA fragments are immobilized on defined regions on a carrier. Such DNA arrays include, for example, those called DNA chips. A gene or a DNA fragment derived from a gene, which is fixed on a carrier at a high density, is also called a DNA microarray. The carrier used for the DNA array may be any carrier that can be used for hybridization, and examples thereof include a slide glass, a silicon chip, an etrocellulose, and a nylon membrane. The shape is not particularly limited, and may be appropriately selected from a flat plate, a disk, a tape, a string and the like.

The method for producing the functional DNA array is not particularly limited.For example, cDNA of a gene having a difference in the base sequence or expression level between the comparison subjects by a differential display method, a subtraction method, an RFLP method ゃ PCR_S SCP method. A library may be prepared, and a DNA selected from the library, preferably a plurality of DNAs, may be immobilized to prepare a functional DNA array. By using this functional DNA array, it is possible to selectively use genes having a difference in base sequence or expression level between comparison targets. In addition, unlike conventional plain arrays, it does not require sequence information or functional information of DNA to be immobilized, which may lead to discovery of new genes.

As the above-mentioned comparison target, for example, two or more types of cells or tissues can be used, and a gene having a difference in base sequence or expression level between the two can be selected. These cells and tissues are not particularly limited to the present invention. For example, they have different origins (derived from different species, derived from different individuals of the same species). (Or different collection sites from the same individual). Two or more types of cells or tissues can be compared. It is also possible to select a gene that has a difference in expression level between a cell or tissue derived from a lesion site or an artificially treated cell or tissue and an appropriate control. As the above controls, the normal site can be used for the lesion site, and the untreated one can be used for those that have been artificially treated. It may be. Examples of the above-described artificial treatment include drug treatment and physical stimulation.

However, when selecting genes whose expression levels differ between the comparison subjects, the differential shazo lady spray method and the subtraction method are both cumbersome, have low reproducibility, and have a large number of false positive clones. However, in the present invention, it is preferable to apply a more sophisticated functional D NAT ray manufacturing technology. In the present invention, DNA microbead array technology such as DNA microphone mouth bead array technology developed by Lynx (U.S. Patent Nos. The functional DNA array obtained by using the technology has the highest performance, and a functional DNA array produced using the technology can be used.

A DNA microbead array (DMA) refers to a collection of various types of microbeads that bind specific DNA. One type of DNA is bound to one microphone mouth bead, and a large number of microbead aggregates form a DNA library. An example of the preparation of DMA is shown below.

First, synthesize anti-tag DNA on microbeads. Each microphone mouth bead has a single-sequence anti-tag, and the anti-tag DNA is a 32-base single-stranded DNA linked to eight 4-base “words”. Each word is one of eight base sequences containing one guanine (G) and three adenine (A) or thymine (T), with three bases differing between any two types of code. Designed. In this way, the 8 ⁸ = 16, 777, 216 types of anti-tags with micro beads is prepared.

On the other hand, 16,777,216 incorporating a sequence (tag) complementary to the anti-tag A cDNA library is prepared using a mixture of different plasmids as vectors. Here, it is designed so that a tag is added to the 3 'end of the cDNA sense strand. After amplifying the tagged cDNA mixture and fluorescent labeling, the tag part is made single-stranded. Mix the above single-stranded tag-bound cDNA and anti-tagged microphone-mouth beads and perform hybridization under stringent conditions. In this process, only completely complementary tags and antitags will hybridize, and combinations of tags and antitags containing at least one different word will not hybridize. The microbeads to which the cDNA is bound are collected by a cell sorter, and the cDNA and the anti-tag are ligated with DNA polymerase and DNA ligase, whereby DMA, which is an aggregate of microbeads in which the cDNA is immobilized by covalent bond, is obtained.

Perform the following procedure to analyze the differences in gene expression between the two types of cells or tissues. Here, a normal part and a cancerous part of the removed organ will be described as an example.

Extracted from normal part and cancer part; cDNA is synthesized using mRNA as type III, and DMA is prepared by the above method. The cDNA bound to the microbeads is subjected to single-strand treatment with cDNA, and equal amounts of the normal part and the cancer part DMA are mixed.

On the other hand, probes labeled with different fluorescent dyes, for example, Cy5 and 6-carboxyfluorescein (FAM), are synthesized based on mRNAs extracted from the normal part and the cancerous part. These are mixed and hybridized with the single-stranded cDNA binding microbeads described above. Thereafter, the fluorescent dye bound to the microbeads is observed with a cell sorter, and the microbeads in which the ratio of the fluorescence of Cy5 to the fluorescence of FAM is out of the preset range are separated into individual containers one by one. A single fragment of a gene having a difference in the expression level between the normal part and the cancerous part can be obtained by performing gene amplification using these microphone-mouth beads as a type III. If necessary, these DNA fragments can be used for cloning into a plasmid vector, nucleotide sequence analysis, probes for cloning full-length cDNA or genomic DNA, and the like.

By immobilizing the gene fragments thus obtained, it is possible to prepare a DNA array in which gene fragments having different expression levels between the normal part and the cancer part are immobilized. When gene expression analysis is performed between normal tissues and cancer tissues, normal cells and cancer cells, and the like using the DNA array, it is expected that differences in expression levels can be observed in many spots. By using the DNA array obtained in this way, the expression level can be reduced by a DNA array with a smaller number of spots compared to a DNA array in which DNA selected by other methods other than DMA technology is immobilized. As a result, it is possible to reduce the cost of DNA array production and the time required for production.

In the above, the normal part and the cancerous part of the removed organ have been described as examples.However, in addition to these, there are various combinations such as cells that have not been treated with drugs, tissues that have been subjected to physical stimulation, and tissues that have not been subjected to physical stimulation. Genes with different expression levels can be easily picked up using DMA technology. As described above, in order to obtain genes having different expression levels, information on the sequence and function is unnecessary, and new genes can be discovered. The DNA array in which the genes thus picked up are immobilized is a DNA array in which only genes related to specific physiological functions are immobilized, that is, a functional DNA array.

The method for selecting genes having a difference in nucleotide sequence is not particularly limited, and known gene polymorphism analysis techniques such as the RFLP method and the PCR-SSCP method can be used. The functional DNA array of the present invention can be obtained by using the gene fragment in which the difference is detected. In addition, using known SNP detection methods, SN

The functional DNA array of the present invention for detecting SNP can also be used using the DNA fragment from which P has been detected. In the DNA array for SNP detection, nucleotides containing SNP, and oligonucleotides can be preferably used in terms of detection sensitivity.

For example, there is no particular limitation on a method for producing a functional DNA array using a functional DNA selected by using the DMA technique, and an optimal functional DNA array may be produced using a known method. That is, cDNA, genomic DNA and their fragments obtained based on the above DNA fragment or the base sequence of this fragment are prepared, and these are immobilized on a suitable carrier to produce a functional D NAT layer. can do. It should be noted that the functional DNA array of the present invention is not limited to those in which all of the genes and their fragments selected as described above are immobilized. The present invention also includes those in which only a part of the selected gene or a fragment thereof is immobilized.

This function1.In a raw DNA array, it is only necessary that DNA is aligned and fixed in a predetermined region on the surface of the carrier, and there is no particular limitation on DNA. A double-stranded polynucleotide having a chain length of 50 bases or more, a single-stranded polynucleotide or a derivative thereof is used. The DNA is prepared by enzymatic amplification based on information of the selected functional DNA by a gene amplification reaction, for example, a gene amplification reaction such as the ICAN method described in WO 00/56877 pamphlet. May be immobilized on a carrier. As the DNA derivative, for example, those modified so as to enable immobilization on the surface of a carrier can be used.Examples of the derivative include, but are not limited to, an amino group at the 5 ′ end of the DNA. DN DNA into which a functional group such as a thiol group is introduced.

The carrier used in the production of the DNA array is not particularly limited, but is preferably a non-porous material having a structure with a smooth surface.For example, glass such as a slide glass can be suitably used. . The surface of the carrier may be any as long as it can immobilize DNA by covalent or non-covalent bonds. In addition, the DNA array may be in the form of a disk or a tape, and the density of the DNA on the carrier may be set under the optimum conditions by the user.

The consortium in the system of the present invention is named a functional DNA array consortium (Function DNA Array DNA Consortium: FDAC).

In the system of the present invention, the FDAC power member can analyze the function of the test gene using the functional DNA array.

In the system of the present invention, the founding company and / or individual establishes a consortium and recruits members, and consortium members request a functional DNA array development company through the consortium to develop the array. By doing so, not only can the cost of developing the array be reduced, but also the development can be streamlined and many results can be obtained in a short time.

In other words, with the system of the present invention, research and development through gene function analysis is not limited to pharmaceutical companies and chemical companies, but a wide range of researchers and R & D companies can select gene analysis projects, and profit It becomes possible to enjoy. In other words, consortium members can manufacture and request various types of functional DNA arrays at lower cost. Research can contribute to raising the level of research and development.

In the present invention, the consortium is funded by a consortium member, and the consortium, upon request of the consortium member, obtains functional D based on information on a specific test gene and Z or a sample containing the test gene. A functional DNA array production company was asked to produce a functional DNA array by sorting the test genes by function and using the genes sorted by function. Provide information on specific test genes and samples containing Z or test genes, order DNA arrays, and provide development funding to DNA array developers.

For each specific DNA array development project, the functional DNA array development company sorted the test genes by function and manufactured and manufactured a functional DNA array using the genes sorted by that function. D NAT Ray will be exclusively delivered to the consortium.

The consortium can distribute the delivered DNA array to consortium members and, if necessary, apply for a patent for the DNA used for the functional DNA array or the DNA array.

The distribution of the functional DNA array delivered to the consortium is at the consortium's discretion, such as free or paid distribution to all members, free or paid distribution according to the investment in the consortium, etc. It can be determined as needed.

Consortium members conduct functional analysis using the functional DNA arrays distributed to them, for example, using the functional DNA arrays for drug efficacy analysis, safety testing, drug screening, etc., and obtained results. You can apply for a patent if necessary.

As an embodiment of the development system of the present invention, there is a development system in which progress information of a functional DNA array development project is disclosed to a consortium regularly or as needed, and the consortium and consortium members develop the function You can get information about the progress of the project. Information on the project and Z or its progress information can be obtained from the -May be disclosed to Members and Consortium Members regularly or as needed. In addition, as an aspect of the development system of the present invention, the right to join the consortium can be bought and sold in a functional DNA array trading market established by the consortium. In the present invention, for example, investment is made through an information communication system. Homes can buy or sell rights. There is a computer system as the information communication system, and the Internet is exemplified as the computer system. A functional DNA array development project is a DNA array development project for gene expression information analysis.

In the present invention, the consortium will open a computer system, especially a functional DNA array trading market via the Internet as needed. Functional DNA arrays can be freely traded in such markets. According to the present invention, a researcher / developer can select a specific functional DNA array from one or more functional D NAT layers presented by the consortium, and the consortium trades in the functional DNA array trading market established by the consortium. You can also benefit from this.

Some aspects of the development system of the present invention have the following requirements.

Legal entities or individuals who are members of the consortium will provide the consortium with information on specific test genes and samples containing Z or test genes as necessary. The consortium requests a functional DNA array development company to develop the array, and the developer will develop a functional DNA array and deliver the functional DNA array exclusively to the consortium. Therefore, consortium members will be able to obtain a wide variety of functional DNA arrays with inexpensive funds.

FIG. 1 is a diagram schematically showing a functional DNA array development system as an example of a functional DNA array development system of the present invention.

Consortium members, such as companies AD, provide samples and funding to the consortium.

The consortium will provide samples provided by members to the functional DNA array developer, order DNA funds, and provide funding.

The DNA array developer develops the DNA array using the optimal functional DNA lay manufacturing technology, for example, the DNA micro-bead array technology, and for example, removes unknown genes. Select, classify, sort, obtain functional genes, create functional DNA arrays, and provide them exclusively to the consortium.

FIG. 2 shows a flowchart of the development system of the present invention. That is, FIG. 2 is a diagram showing a functional DNA chip development system as one embodiment of the functional DNA array development system of the present invention.

Each member of the consortium will provide the consortium with the necessary samples and funding to produce the desired functional DNA chip.

The consortium will provide samples and funds requested by each company to the functional DNA chip development company, and place an order for functional DNA chips.

The functional DNA chip developer sorts genes with different expression levels between, for example, two types of cells or tissues using, for example, DNA microphone array beads array technology, and fixes the sorted genes or their fragments. Prepare a DNA chip on which the immobilized gene of unknown sequence is immobilized. If necessary, the sequence of the unknown gene is analyzed.

The manufactured functional DNA chip is exclusively provided to the consortium, distributed from the consortium to each company that is a member of the consortium according to the purpose, and each company obtains the research and development results obtained using the DNA chip for each function. Make money with. One embodiment of the functional DNA array development system of the present invention is exemplified below. In the functional DNA array development system of the present invention, the consortium created by the promoter authorizes consortium members to use a functional DNA array corresponding to a pathology created by, for example, a functional DNA array development company. be able to.

With the funding of the consortium members, the consortium will be able to pay the development costs for each functional DNA array to the functional DNA array developer.

Consortium members can use the provided functional DNA arrays (for example, functional DNA arrays according to pathology) to independently develop drugs, etc., and can outsource research to third parties. The results can be obtained independently.

The consortium itself is not particularly limited to possessing the functions of a public institution or an academic institution (academic center). It is only necessary to have the function of ordering a functional DNA array from a functional DNA array developer and providing the manufactured array to consortium members.

By using the provided functional DNA array, consortium members will be able to start research and development using the array in the same position and with the same chance. The results can be obtained independently.

A functional DNA array developed by a functional DNA array developer, for example, describes only pathological classifications, is distributed to desired members, and is used for DNA analysis. Will be performed independently by each consortium member without any restrictions.

For example, functional D using rare disease-related genes in a small number of cases

NA arrays are priced for each functional DNA array based on the relationship between supply and demand, such as making the cost lower than that of a functional DNA array using genes related to diseases with a large number of cases, for example, cardiac teratology. Can be set.

The consortium may collect pathological tissues and healthy tissues used for the preparation of functional DNA arrays from the polentia tissue, medical science network, gene center, etc.

Collectibles may also be collected from consortium powers [[members] and may be collected via the consortium at collection centers.

The agglomeration center may be established by the consortium or may be independent of the consortium members. The accumulation center manages the quality of collected samples, for example, pathological tissues and healthy tissues, and at the request of the consortium, prepares samples for the development of functional DNA arrays according to the business situation of the functional DNA array development company. You can send it.

Through these means, the consortium can contribute to the promotion of human health science without the functions and costs of an academic or medical science research organization. As described above, according to the present invention, the founder creates a consortium, recruits consortium members, for example, provides funds to the consortium by companies and individuals, provides gene samples to the consortium, and functions of the sample from the consortium 'I4D NA array Provision to development company, ordering of functional DNA array, DNA array development meeting Optimal functional DNA array production technology by the company, e.g., DNA array production using the DNA microarray bead array technology, e.g., selection, classification, and sorting of unknown genes, and functions obtained by acquiring functional genes A functional DNA array development system will be provided that includes the delivery of a functional DNA array to the consortium, the distribution of the array from the consortium to consortium members, and the research and development of the array by the consortium members.

The system of the present invention allows consortium members to request the functional DNA array development company through the consortium for the development of such arrays, thereby reducing the costs required for the development of functional DNA arrays required by consortium members. Not only can you save money, you can also streamline development and get more results in less time.

In other words, the system of the present invention enables research and development through gene function analysis not only to pharmaceutical companies and chemical companies with large capital, but also to various researchers and R & D companies, and becomes a consortium member. Would be able to select a gene analysis project under the same conditions and enjoy the benefits of R & D.

The system of the present invention allows consortium members to obtain various types of functional DNA arrays at a lower cost and easier access. It is extremely useful for raising the level of

Example

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of DNA microbead array and selection of genes

Proc. Natl. Acad. Sci. USA, Proc. Natl. Acad. Sci. USA, Vol. 97, pp. 1665-167

According to the method described on page 0, (2000) (hereinafter referred to as Reference 1), a DNA microbead array was prepared as shown below.

(1) cDNA synthesis

Human promyelocytic leukemia cells HL-60 cells (ATCC CCL-240) Suspended in RPMI 1640 medium (manufactured by BioWhitaka Inc.) containing 0% fetal bovine serum (manufactured by JRH Biosciences) and 1.3% dimethyl sulfoxide, respectively, and incubated with 37% in the presence of 5% carbon dioxide. After culturing for a week, the cells were induced to differentiate into neutrophils. The cells obtained were collected by centrifugation, suspended in RPM11640 medium containing 10% fetal bovine serum to a concentration of 1 × 10 ⁶ eel 1 s / m1, and the suspension was placed in a 10 cm Petri dish. 1 Oml was added to each of the two plates. 100 µg of water (Α) in one dish and 100 µg / m 1 honolepole myristate acetate (TPA, Gibbone clay) dimethylsulfoxide solution (B) in the other dish Each was added and cultured for 4 hours. All cells from (A) and (B) collected from each Petri dish were treated with Trizol reagent (Gibco).

RNA was prepared, and then mRNA was purified from total RNA using Oligotex τ ^M _dT30 (Takara Shuzo). Using this mRNA 2.5 // g as type II, cDNA was synthesized using a cDNA synthesis kit (Stratagene). At this time, a 5 ′ biotin-labeled primer having the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing was used as a primer for reverse transcription.

The cDNA thus obtained was treated with phenol-form and subjected to gel filtration and purification using Sepharose CL4B (manufactured by Amersham Pharmacia), followed by ethanol precipitation. The cDNA recovered as a precipitate was dissolved in 200/1 DpnII buffer (New England Biolabs) and digested with the restriction enzyme DpnII (New England Biolabs). After recovering the 3'-side portion of the cDNA from the digest using streptavidin magnet beads (manufactured by Dynal), add 20 1 of 10XNEB # 2 buffer (manufactured by New England Biolabs) to a total volume of 200 ml. It was adjusted to 1 and digested with the restriction enzyme B smB I (New England Biolabs). The reaction solution was subjected to phenol-form mouth treatment and ethanol precipitation, and the precipitate was dissolved in sterilized water 10.51, and this was used as a 3 'side cDNA solution in the following experiments.

(2) Hybridization of the side cDNA and anti-tagged microbeads Example 1 1 μl of the 3′-side cDNA solution derived from (A) and (B) synthesized in (1) was inserted into 200 ng of the tag vector pLCV2 described in Reference 1 to obtain 140,000 to 18 Six plasmid pools consisting of 10,000 clones were prepared. One 100 μl reaction mixture of 5 ′ biotin-labeled primer of the nucleotide sequence shown in SEQ ID No. 2 of the sequence listing and 5 ′ FAM-labeled primer of the nucleotide sequence shown in SEQ ID No. 3 of the sequence listing was added. For each plasmid pool, 100 PCRs were used, and PCR was performed using 125 ng of each of the above-mentioned brasmid pools per type as one reaction solution. PCR was performed under the following conditions.

94. C—hold for 3 minutes

8 cycles of 1 cycle at 94 ° C-30 seconds, 67 ° C_3 minutes

22 cycles of 1 cycle of 94 ° C-30 seconds, 64 ° C-3 minutes

6 7 ° C—hold for 3 minutes

After the completion of the reaction, the PCR reaction solution was collected into a single tube for each pool, subjected to phenolic-form-form processing and ethanol precipitation, and then the obtained precipitate was dissolved in sterilized water (1200 / z1). After adding 180 μl of 10 XNEB # 4 buffer (manufactured by New England Biolabs) to make the total volume 1800 μl, digestion with the restriction enzyme PacI (manufactured by New England Biolabs) was performed. . After removing the fraction adsorbed by Ultralink SA Resin (Pierce) from the reaction solution, the cDNA solution obtained is subjected to phenol-form mouth treatment and ethanol precipitation. Dissolved. Here, 180 μl of 10 XNEB # 2 buffer solution (manufactured by New England Biolabs) was charged, and the total amount was adjusted to 1800 μl, followed by treatment with Τ4 DNA polymerase (manufactured by Takara Shuzo). At this time, the single-stranded chain of the tag was added by adding only dGT GT as a substrate. 300 μg of each pool of the cDNA having the single-stranded tag was mixed with 144 million antimicrobial microphone-tagged beads described in Reference 1, and 400 μl of hybridization buffer (1 OmM phosphate buffer) was mixed. The solution (pH 7.2), 0.5 M NaCl, 0.01% Tween 20, 1.2% dextran sulfate) was hybridized for 3 days with shaking at 72 ° C. The microbeads were collected by centrifugation and 500/1 of 10111] \ [Tris-HC1 (pH 7. 9), 1 OmM Mg C 1 2, 5 OmM N a C 1, 1 mM Jichiosureito Le, 0. 01% Twe KoTsuta the shaking washed 30 minutes. 64 to 70 ° C and suspended in en 20. After repeating this washing operation several times, 6 pooled microbeads with washed cDNA were collected and MoF 1 was added. Measure the FAM fluorescence intensity (excitation wavelength: 488 nm, detection wavelength: 530 nm) of the beads of the microphone mouth using a cytometer (manufactured by Cytomation). The beads were collected. By the above operation, about 6 million microphone mouth beads to which cDNA was bound by hybridization were obtained.

(3) Preparation of DNA microbead suspension

Example 11 About 2 million of the microbeads obtained by hybridization in which the cDNA was obtained by hybridization in Example 11 (2) were used for 100/1 of 10 mM Tris-HC1 (pH 7.9), _{10 mM M g C 1 2,} 50 mM N a C 1, 1. 4m M dithiothreitol one Honoré, 0. 01% Twe en 20, each 0. 1 mM of d a TP, dGTP, dTTP, 5- methyl dCTP, Suspend in 1 mM ATP and add 2 OU T4 DNA polymerase and 40 OU Τ4 DNA ligase.

The reaction was performed at 12 ° C for 2 hours. The reaction was stopped by pronase treatment, and the microbeads were collected from the reaction solution by centrifugation, and 100 μl of DpnII buffer was added.

(New England Biolabs) and digested with Dpn II (New England Biolabs). The microbeads were collected by centrifugation and 100 // 1 1 OmM Tris-HC1 (pH 7.5), 5 mM

Suspend in MgCl ₂ , 0.033 mM dGTP, 7.5 mM dithiothreitol, 0.01% Tween 20, and add 10 U of DNA polymerase I large fragment (Takara Shuzo) at 25 ° C for 15 minutes. Reacted. Microbeads were collected by centrifugation, suspended in ligation buffer (manufactured by Takara Shuzo Co., Ltd.), and DNA of the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 in the Sequence Listing was aerial in advance. The prepared adapter (0.1 nmol) was added, and a ligation reaction was performed using # 4 DNA ligase (Takara Shuzo). The microbeads were collected by centrifugation, suspended in a bead stripping solution (15 OmM NaOH, 0.01% Tween 20), and reacted at room temperature for 10 minutes. Farther than the reaction solution After removing the supernatant by centrifugation, the microbeads were washed with 1 OmM Tris-HC 1 (pH 8.0), ImM EDTA, 0.01% Tween 20, and 1 ml of l OmM Tris-HC 1 ( pH 8.0), ImM EDTA, 0.

Suspension was performed in 01% Tween 20 to obtain a DNA bead suspension from Sample No. 1 and Sample 2.

(4) Preparation of probe

Example 1—The 3′-side cDNA li 1 derived from (A) and (B) prepared in (1) was inserted into 200 ng of the probe vector p LCV described in Reference 1, respectively, to obtain from 800,000 to 10 million clones. Was prepared. After adding universal buffer 20 (manufactured by Takara Shuzo) 20 // 1 to each 10 μg of this plasmid to make the total volume 200 μ1, then use the restriction enzyme S au3AI (manufactured by Takara Shuzo) to delete the plasmid. did.

(A) 1.2 μg of the Sau3AI digested plasmid derived from (A) was used as a type II protein using the nucleotide sequence shown in SEQ ID NO: 6, 5, FAM-labeled primer 2 nmo1, and

(B) 2.4 μg of Sau3AI-digested plasmid derived from type B, using a 5 nm Cy5 labeled primer 4 nmo1 of the nucleotide sequence shown in SEQ ID NO: 7 in the sequence listing I did a Reyua PC R. Lyure PCR was performed under the following conditions.

95 ⁰ C—hold for 5 minutes

25 cycles with one cycle of 95 ° C-30 seconds, 64 ° C-30 seconds, 72 ° C-30 seconds

Hold at 72 ° C for 10 minutes _ Purify the amplified DNA from each of the reaction solutions after the rear-air PCR using the QiaQuick PCR Piperification Kit (Qiagen), precipitate with ethanol, and then use sterile water 21 μl. The sample was dissolved in 1 to obtain a FAM fluorescently labeled probe derived from sample 1 and a Cy5 fluorescently labeled probe derived from sample 2.

(5) Hybridization with DNA microbeads

Example 1 DNA microbeads derived from sample 1 prepared in (3) and DNA microbeads derived from sample 2 were mixed in an amount of about 360,000 each, and this bead was mixed with sample 1 derived from sample 1 prepared in (4). Add 5 g of each of the Cy5 fluorescently labeled probes derived from FAM fluorescently labeled Puyopu sample 2 and add 50 μl of buffer (4 XSS Hybridization was carried out overnight at 65 ° C with shaking in C, 25% honolemamide, 0.1% SDS). The microbeads collected by centrifugation were washed with 1X SSC, 0.1% SDS solution at 65 ° C for 30 minutes, and then washed with 0.1 XSSC, 0.1% SDS at 65 for 30 minutes, Collect the DNA microbead beads by centrifugation, and mix them with 10 mM Tris-HC1 (pH 8.0), 1 mM EDTA, 0.

The suspension was suspended in 01% Tween 20 to obtain hybridized DNA microphone bead beads.

(6) Sorting

The hybridized DNA microbeads obtained in Example 11- (5) were subjected to sorting using a MoF 1 o cytometer (manufactured by Cytomation). Measure the fluorescence intensity of FAM (excitation wavelength: 488 nm, detection wavelength: 530 nm) and the fluorescence intensity of Cy5 (excitation wavelength: 647 nm, detection wavelength: 670 nm) of the DNA microbeads. 3692 DNA microbeads with high fluorescence intensity (bead suspension A) and 3349 DNA microbeads with strong Cy5 fluorescence intensity (bead suspension B) were fractionated.

(7) PCR and DNA nucleotide sequencing

Example 11 PCR was performed on the bead suspensions A and B fractionated in Example 1 (6) using the primers whose base sequences were shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively, in Type I. To 100 μl of the reaction solution, 200 铸 -type beads and 40 pmol of the primer were added, and five reactions were performed for each of the bead suspensions A and B. PCR was performed under the following conditions.

95 ° C—hold for 5 minutes

25 cycles with 1 cycle of 95 ° C-30 seconds, 64 ° C-30 seconds, 72 ° C_30 seconds

72 ° C—hold for 10 minutes

Combine the PCR reaction solution into one for each of suspensions A and B, mix 1 μl of each with Clawing vector ρ Τ 7 Β 1 ue (manufactured by Novagen), ligate and mix. , Plasmids were purified from a total of 1920 colonies, 960 colonies from bead suspension A and 960 colonies from bead suspension B. The DNA sequence of the DNA fragment was analyzed. Among the obtained base sequences, those that satisfy one of the following conditions were selected.

Condition 1: The length of the insert rooster is 40 bases or more.

Condition 2: The length of the insert sequence is 30 bases or more and 40 bases or less and does not have a po1yA sequence.

When the same insert sequence appeared in a plurality of plasmids, one of the plasmids was selected, and 740 plasmids were obtained as a type I plasmid for DNA microarray.

Example 2 Preparation of DNA microarray

Using the 740 plasmids obtained in Example 1 as type III, PCR was performed using a primer pair having the nucleotide sequence shown in SEQ ID NOS: 10 and 11 in the sequence listing. Then, the obtained amplification product is subjected to ethanol precipitation, and the obtained precipitate is dissolved in lXSolution T (manufactured by Takara Shuzo Co., Ltd.) to a concentration of 0.3 μl, and this is dissolved in a spotting solution used for making an array. And

The spotting solution was spotted on MAS-coated slide glass (Matsunami Glass Co., Ltd.), which is an aminated slide glass, using Affymetrix 417 Arrayer (Affy Metrix). 3 glass slides after spotting. After maintaining at C ヽ humidity 90% for 1 hour, UV irradiation of 60mjZcm ² was performed to perform crosslink. Further, after the slide glass was washed once with 0.2% SDS and then twice with distilled water, 2.75 g of succinic anhydride and 162.5 ml of N-methyl_2- Pyrrolidone was immersed in 15 ml of a 1 M borate buffer (pH 8.0) viable blocking solution for 20 minutes to block free amino groups. Next, the slide glass was washed twice with distilled water, heat-denatured in boiling water for 2 minutes, and quenched with 100% ethanol at ice temperature. Thereafter, the slide glass was dried by low-speed centrifugation and used as a DNA microarray in the following experiments.

Example 3 Cell Preparation

HL-60 cells were suspended in RPMI 1640 medium containing 10% fetal serum (manufactured by JRH Biosciences) and 1.3% dimethyl sulfoxide, and then suspended in the presence of 5% carbon dioxide. The cells were cultured for 1 week, and the cells were induced to differentiate into neutrophils. The cells obtained were collected by centrifugation, suspended in RPMI 1640 medium containing 10% fetal calf serum to a concentration of 1 x 10 ⁶ ce 11 s / m1, and the suspension was placed in a 10 cm Petri dish. One Om1 was added, and each was treated as follows: 1 100 μl of 4 mM DGE aqueous solution was added and the RNA was recovered 4 hours after the addition. ② 10 L of 1 Ο Ομg ZmL phorbol myristate acetate (TPA, manufactured by Gipcone earth) A section in which RNA was recovered 4 hours after the addition of the dimethyl sulfoxide solution. ③ After addition of 4 mL of 4 mM aqueous DGE solution and culturing for 6 hours, an additional 10 μL of 100 μg; mL TPA The category in which RNA was collected 4 hours after adding the dimethyl sulfoxide solution, and the category in which RNA was collected 4 hours after adding 100 L of water as a negative control.

Example 4 Expression analysis using DNA microarray

(1) Preparation of labeled cDNA for detection and hybridization

From each of the HL-60 cells prepared in Example 3, using Trizo 1 reagent and O 1 igote x-dT30 <Supper> (manufactured by Takara Shuzo Co., Ltd.), according to the protocol of each kit, Po 1 y A (+) RNA was prepared. Next, 1.0 μg of Po1yA (+) RNA was used as type III, and the kit was prepared using RNA F1uorescence Labeling Core Kit (M-MLV Version, manufactured by Takara Shuzo). According to the protocol, cDNA labeled with Cy 3 and Cy 5 respectively was prepared. The obtained labeled cDNAs were mixed in the combinations shown in Table 1 to prepare samples used in the following experiments.

In accordance with the operating procedure described in the instruction manual for the Inte 11 iGene (Takara Shuzo), the DNA microarray prepared in Example 2 was mixed with the Cy3-labeled cDNA and Cy5 label. Hybridization with each of the above samples 1 to 4 was performed, and the operations from washing to drying were performed. Then, Cy3 (excitation wavelength: 532 nm, detection wavelength: 570 nm) and Cy5 (excitation wavelength: 635 nm) were used using A Af yme tri X 428 Ar ray Scanner (manufactured by Affimetrix). _s detection wavelength: 660 nm). Then, the average value of the signal intensity of the spot area of each spot and the average value and the standard deviation of the signal intensity of the area around the spot (hereinafter referred to as the background area) were calculated using the image quantitative analysis software ImaGen 4.1 ( (Manufactured by Biodiscovery).

(2) Gnorape of each clone

To group each clone by statistical analysis based on the gene expression ratio, it is necessary to remove data of low reliability and data of clones showing no significant variation. Therefore, clones to be subjected to groupi dang were narrowed down by the following procedure. That is, from the average value and the standard deviation of the signal intensity in the background region obtained in Example 4 (1), a signal intensity threshold was obtained by the following formula.

Signal intensity threshold = average signal intensity in background + 2 X

(Standard Deviation of Signal Intensity of Region) The significance of the spot signal in each channel was determined based on whether or not the average value of the signal intensity of each spot region was larger than the above threshold. Then, in all four samples, clones in spots with no effective signal on both the Cy3 and Cy5 channels were excluded. Furthermore, the ratio of the Cy 5 signal intensity to the Cy 3 signal (hereinafter referred to as the expression ratio) was calculated, and for all the clones, the ratio was larger than 1/2 and smaller than 2 in all samples. Clones that did not show a change were excluded from further analysis.

As a result of the above refinement, 227 clones were selected from 740 clones. Was selected.

Next, clustering between clones was performed from the expression ratio data by the following procedure.

That is, out of the above-mentioned 227 clones, clones in which the average value of the signal intensity of the spot was greater than the average value of the background signal intensity in all four sample sumps were extracted, and a total of 124 clones were extracted. Was selected. In addition, clones that were insignificant in both the Cy3 and Cy5 signals in any of the 4 samples were removed, and a total of 82 clones were finally selected. Thereafter, the logarithm of the expression ratio with the base at 2 was determined (hereinafter referred to as the Log 2 ratio). Next, the correlation coefficient between the four clones of the data of the Log 2 ratio was obtained, and hierarchical clustering was performed using the average distance method as a combination method using the correlation coefficient as a similarity. From the obtained dendrograms, clusters with a correlation coefficient of 0.95 or more were classified into one group. As a result of the above clustering, the above-mentioned grouping of 82 clones resulted in a total of 18 groups. When the base sequence of each clone contained in each group was searched for homology against the gene database using the computer algorithm BLAST, it was found that there was no homology with the known gene and that a new gene was expected. And 75 corresponding genes existed. As described above, seven novel genes and 72 known genes related to the mechanism of action of TPA and / or DGE on the living body were obtained. Industrial applicability

In the functional DNA array of the present invention, genes sorted by function or their fragments are immobilized. By using the array, gene expression analysis by function, which cannot be performed by the conventional plain array, can be easily performed. That is, the array of the present invention is extremely useful for conducting comprehensive and detailed research on the functions of living organisms involving genes immobilized thereon. In addition, by using a DNA microbead array in its production, it is possible to efficiently and functionally produce a functionally-generated DNA array without overlooking useful genes.

Claims

The scope of the claims

1. A DNA array in which one or more genes or fragments thereof selected as having a difference in the base sequence or expression level between the comparison targets are fixed to a predetermined region on a carrier.

2. The DNA array according to claim 1, wherein the comparison target is two or more types of cells or tissues.

3. The DNA array according to claim 2, wherein the comparison target is two or more cells or tissues of different origins.

4. The DNA array according to claim 3, wherein at least one of the comparison targets is a cell or tissue derived from a lesion site.

5. The DNA array according to claim 3, wherein at least one of the comparison targets is an artificially treated cell or tissue.

6. A method for producing a DN array according to claim 1, wherein

(1) a step of selecting a gene having a different base sequence or its expression level among the comparison targets,

(2) a step of preparing the gene selected in (1) or a fragment thereof, and

(3) A method for producing a functional DNA array, comprising a step of immobilizing the gene or the fragment thereof prepared in (2) above on a predetermined region on a carrier.

7. The method for producing a functional DNA array according to claim 6, wherein step (1) is performed using a DNA microbead array.

8. Function: raw DNA array development system,

The consortium founded by the founders recruits consortium members, raises funds from consortium members,

The consortium orders a functional DNA array developer to produce a functional DNA array and provides information on specific test genes and samples containing Z or test genes to the functional DNA array developer as needed And

The functional DNA array developer sorts the test genes by function to create a functional DNA array, and uses the functional DNA that is sorted by the function Manufacture arrays and provide them exclusively to the consortium,

Functional DNA array development system characterized by the following features:

9. The functional DNA array development system according to claim 8, wherein the functional DNA array is a DNA array for analyzing gene expression information.