JP2002507275A - How to make a subtraction library - Google Patents

Abstract

  (57) [Summary] A method for making a subtraction library using a defined collection of sequences is described. The method comprises providing a surface containing a collection of known nucleic acid sequences, and then contacting the surface with a library containing undefined sequences under appropriate hybridization conditions. Unhybridized DNA is recovered and sequenced. The sequenced unhybridized DNA forms a subtraction library, which contains sequences that were present in the library and that differ from the sequences of the collection. Also described is a subtraction library produced by the method of the invention. Methods for making and using subtraction probes are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION                       How to make a subtraction library   This application is related to US Provisional Application No. 60/041688 filed March 24, 1997. And claim priority.Field of the invention   The present invention relates generally to the field of cDNA libraries, and more particularly to The present invention relates to a method for manufacturing subtraction libraries.Background of the Invention   Using a so-called "high-density DNA array" or grid Methods for obtaining information about gene expression and identity have been described. An example For example, M. Chee et al., Science, 274: 610-614 (1966) and others cited therein. See literature. Such a gridding assay is based on the expression sequence tag. (Ada) has been used to identify a specific novel gene sequence called EST (Ada). ms et al., Science, 252: 1651-1656 (1991)). Also identified based on gene products Various methods for identifying the gene sequence of have also been described. For example, 1991 See International Patent Application WO 91/07087, published May 30, 1980. In addition, the desired sequence Are also described. For example, an international patent published on November 14, 1991 See application WO 91/07271.   Currently available subtraction methods are not desirable from a given array. This is to remove the wrong sequence. One approach uses many unknown genes Drive the subtraction library to remove unknown genes from the target library I do. For example, J. Love and P. Deininger, BioTechniques, 11 (1): 88-92 (1991) reference. However, this method is useful for removing large numbers of genes from a library. For the genes in the resulting subtraction library, Except for Little is known. One commonly used approach for performing subtraction Dry subtraction using a small number of known genes (eg, less than 100) To remove these genes from the library of interest. This approach Control the removal of some genes, but current methods do not The roach can only be used for a small number of genes and the resulting draw Computational libraries contain significant amounts of unwanted genes.   Therefore, there is a need for a more efficient method for the production of subtraction libraries. There is a need. Also, a more efficient method for screening new pharmaceutical reagents Is also necessary.Summary of the Invention   In one embodiment, the present invention relates to a collection comprising a known or defined sequence. The present invention provides a method for manufacturing a subtraction library by using a method. The method is defined Providing a surface having a collection of isolated nucleic acid sequences, followed by hybridization. Library containing undefined nucleic acid sequences under conditions that allow Associating with the surface of For nucleic acid sequences that do not hybridize Harvest, isolate, and then create a subtraction library. The first of the defined array Undefined sequences from a second library that are not present in the library To characterize the subtraction library.   In another aspect, the invention relates to a subtraction line produced by the method of the invention. Provide a blurry.   In yet another aspect, the present invention provides a method comprising using the method steps described herein, If there is a known or defined sequence, Provided is a method for rapidly screening an library.   In yet another aspect, the present invention provides a method comprising using the method steps described herein, Defined using polynucleotide probes from known or defined sequences To rapidly screen libraries containing unselected sequences You.   Other aspects and advantages of the present invention are described below in preferred embodiments of the invention. One This will be further described in the detailed description.Detailed description of the invention   The present invention provides a subtraction line using a collection of known or defined sequences. A method for manufacturing a library is provided. The method may comprise a defined nucleic acid sequence or a known nucleic acid sequence. Provide a surface on which the collection of nucleic acid sequences is immobilized, and then Under hybridization conditions, lines containing unknown or undefined sequences Contacting the surface with a library. Sequences that existed in the library As a subtraction library that contains an array that is different from the collection array Recovering unhybridized polynucleotides, preferably DNA You. Advantageously, the method of the present invention drives subtraction using multiple defined arrays. To be able to Therefore, this method can easily control the contents Enables efficient production of subtraction libraries.   Also described is a subtraction library produced by the method of the invention. I.Definition   Some words and phrases used throughout this specification are defined as follows: Justify.   The term "gene" as used herein refers to the genomic nucleotide sequence from which the cDNA sequence is derived. A column. Classically, the term “gene” refers to a genomic sequence, Can produce different RNAs.   "Gene product" refers to a polypeptide sequence, peptide or other gene encoded by a gene. Or protein.   As used herein, the term "collection containing known sequences" refers to RNA and DNA sequences. Refers to an ordered set of nucleotide sequences, including PCR products, genes, gene fragments, DNA fragments, oligonucleotides It may be in the form of leotide or the like. Preferably, such known arrangements in the collection The sequence has already been sequenced and / or of known origin . Desirably Means that this collection contains a large number of sequences, for example 10,000 if the sequence is a gene. From 0 to 200,000 species or members of the collection In the case of tide, it contains 1,000,000 species. However, the number of sequences of the invention And is not limited by the present invention. This collection Contains sequences from many different sources, including various libraries. Is also good. For example, a collection may be a member of a mammalian species, such as one of a human or May be derived from more tissue sources. Desirably, humans are healthy You. However, using libraries from sick or compromised individuals, You may. In addition, other target mammals include non-human primates, rodents, and others. And dogs, but not limited to. In particularly preferred collections, The defined sequence exists as a single copy.   When a gene sequence is used in the method of the present invention, the full-length sequence of the gene is known. Need not be. Rather, the method of the present invention makes the gene sequence unique It is necessary that the part be known, and that part is about 17 base pairs. "Known `` Nucleic acid sequence '' is one in which the sequence is fairly unique and includes degeneracy or repeats Means no.   The term “library” refers to a plasmid library, an RNA library, DNA libraries such as libraries containing PCR products from nome genes , CDNA libraries, oligonucleotide libraries and known sequences Including, but not limited to. Methods for constructing such libraries are well known to those skilled in the art. Have been. Minimize the number of complete genes present in a single insert to about one The library may be adjusted to be This adjustment method is well known to those skilled in the art. I have.   "Isolated" is altered from its natural state "by the hand of man"; That is, if it occurs in nature, it has been changed or removed from its original environment Or both are being done. For example, its natural A polynucleotide or polypep naturally present in a living animal in a state Pide is not "isolated", but is the same port isolated from its natural coexisting material. A polynucleotide or polypeptide is isolated according to the terminology herein. You. For example, For a nucleotide, the term "isolated" refers to the chromosome or the chromosome where it originally resides. And separated from cells.   As used herein, the term `` solid support '' refers to a plurality of defined materials (from a library). Refers to the substrate used to immobilize The method comprises combining the other polynucleotide in the sample with the immobilized polynucleotide sequence. It allows for detectable hybridization. Many available Among the solid supports, a desirable example is the international support published on May 30, 1991. It is described in application WO 91/07087. Examples of other useful supports are Including nitrocellulose, nylon, glass, silica and Pall BI0DYNE membranes However, it is not limited to these. An improvement of a conventional solid support may be used in the present invention. Conceivable.   The term "grid" generally refers to a two-dimensional structure on a solid support, The material defined in the library is attached or fixed.   As used herein, the term "predetermined area" refers to one or more copies. Regions that are immobilized and localized on the solid support surface as widened gene regions Hybridization of the clone to the sample polynucleotide. If this is the case, the region should allow for hybridization of the clone at that location. U.   "Immobilization" refers to the attachment of a gene or other nucleic acid to a solid support. Fixed The means of formation are known to those skilled in the art, are conventional, and depend on the type of support used. May exist.   As used herein, the term “label” refers to a label that is easily attached to RNA or DNA. A conventional molecule that can produce a detectable signal. The signal intensity is measured by the DNA fragment or oligonucleotide on the grid. The relative amount of RNA hybridization is shown. Preferred labels are fluorescent molecules or Is a radioactive molecule. A variety of well-known labels can be used.   As used herein, the term “subtraction library” refers to a very rich set of novel nucleotide sequences. Refers to enriched libraries, especially novel genes and gene fragments Including II.Collection of known sequences   In carrying out this method, a collection of defined sequences immobilized on a surface Up to one so that each grid carries the nucleotide sequence from the solid on the solid surface. Or a larger grid is prepared. Preferably, this collection is defined above These nucleotide sequences are, for example, genes, gene fragments. , Another DNA fragment, or an oligonucleotide sequence.   The nucleotide sequence from the selected collection is And Desirably, the nucleotides are in the form of DNA, but not otherwise. The nucleotide may be about 100 pg per spot, depending on the base material. Placed on the surface in an amount of about 1000 ng. In a further preferred embodiment, the use The amount of polynucleotide for use is about 10 ng to about 100 ng per spot. . However, similar amounts of RNA may be used. Not necessarily, but gene Or apply other nucleotide sequences on the surface as double or multiple areas And may be desirable. Nucleotide sequence spotted on solid support surface Individual clones grown and expanded; or isolated from the above libraries Plasmid clone, PCR product from plasmid clone, or plasmid Includes (but not limited to) oligonucleotides obtained by sequencing of clones Not), they are immobilized on the solid support surface.   These nucleotide sequences are often immobilized on various solid support surfaces. Is used. For example, Affinity Techniques, Enzyme Purificati on: PartP, Methods in Enzymology, Vol. 34, ed. W. B. Jakoby, M .; Wilcheck Acad. Press, NY (1971); Immobilized Biochemicals and Affinity Chromatog raphy, Advances in Experimental Medicine and Biology, Vol. 42, ed. R. Du nlap, Plenum Press, NY (1974); U.S. Pat. No. 4,768,881; U.S. Pat. No. 42102; EP-A-391608 (October 10, 1990) Or see U.S. Pat. No. 4,992,127 (November 21, 1989).   Although not necessary, the sequence of the gene or other nucleic acid sequence Should be located at predetermined locations or areas on the surface. No. Understanding how the array was platooned was based on prior art methods. A higher level of efficiency is provided to the method of the invention than is achievable in some cases. This means It is an important feature of the present invention that facilitates access to the desired sequence and preferably Facilitate access to related clones and related data after leaning. this One preferred method for attaching these materials to a solid support is described in International Patent Application PC T / US90 / 06607 (published May 30, 1991). Simple To explain simply, this method is used before the material can be immobilized on the surface of the solid support. Forming a predetermined area. The method selects a predetermined area A surface-attached binding substrate is used that allows for an active activation. Activated These substrates then bind and immobilize material from a defined sequence collection. become able to.   Suitable for binding nucleotide sequences on surfaces for hybridization The solid substrate, and the method of attaching the nucleotide sequence to the substrate, are in accordance with the present invention. Those skilled in the art can use it. However, currently the preferred surface is glass. You. For other solid substrates, how to deposit and attach nucleotides to the glass surface The method is well known to those skilled in the art. For example, L. A. Crisey, et al., "Covalent At tachment of Synthetic DNA to Self-Assembled Monolayer Films ", Nucleic Ac ids Res., 24: 3031-3039 (1996); Silicon Compounds: Register and Review (Uni ted Chemical Technologies, Inc., Bristol, Pennsylvania, 1993). Departure In another clear embodiment, the surface may be a bead. The surface is not necessarily flat Since they need not be, vertical surfaces are also considered to be within the scope of the present invention. Such a table The surface may have stairs, ridges, twists, terraces, and the like. III.Libraries containing undefined sequences   Once a grid surface containing immobilized sequences from the collection has been prepared, Under hybridization conditions, libraries containing undefined sequences Can be associated with the original sequence. Such libraries contain sequences of unknown origin and And / or may include a known nucleotide sequence.   Unknown or undefined genes, gene fragments, or other nucleic acid sequences Column Libraries that provide a source of DNA are random cDNA libraries obtained using known methods. It may be an library. Alternatively, a gene from a selected organ or tissue Libraries, or mixed sets of RNA, may be the source of the sequence. Appropriately For use in the method of the present invention, see Sambrook et al., MOLECULAR CLONING, A LABORA. RTORY MANUAL, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring H Using standard methods for molecular biology as disclosed by Arbor, NY 1989 RNA is isolated and reverse transcribed into cDNA. Then, Fleischmann et al., Scienc e, 269: 496-512 (1995). To construct. For purposes of the present invention, a library containing undefined sequences May be a library as described above. Most preferably, the library is This is a plasmid library.   In a preferred embodiment, the source of the unknown or undefined sequence is human DN A, preferably a phagemid library consisting of human cDNA. This tool An example is a hybridizer constituting a subtraction library produced by the method of the present invention. It is particularly advantageous for isolating sequences that are not to be immobilized. Using such conventional methods, A phagemid library can be prepared. For example, the sentence of Sambrook et al. Reference. A suitable vector for this system is pBluescript [Stratagene, L a Jolla, CA] and pUC118 [J. Vieiraand J. Messing, Methods in Enzymolog y, 153: 3 (1987)]. Other suitable vectors are well known to those of skill in the art. , Can be easily selected.   However, as described above, they may be defined using other suitable methods and vectors. Unprepared libraries may be prepared. For example, a vector like M13 May be used to produce a phage library. Other suitable vectors are known in the art. Is known in   These undefined sequences are labeled and hybridized to the immobilized sequence. Alternatively, it may be possible to detect the DNA to be charged. Known idiomatic for labeling sequences A method may be used. For example, fluorescence, radioactivity, photoactivation, biotinylation, energy Energy transfer, solid state circuits and the like may be used in the present invention.   Desirably, libraries containing undefined sequences, using conventional methods Or The single-stranded standard DNA is isolated therefrom. For example, if the library is a phage If so, a single-stranded copy of the library using the appropriate helper phage In a particle. Next, the obtained phage particles are subjected to a conventional method. Although isolated, typically conventional methods include centrifugation and precipitation. . Such methods for the isolation of single-stranded DNA are well known to those skilled in the art and It is not a limiting factor. Although not so desirable, double-stranded DNA May be used in the lighting method. IV.Hybridization to grid   An isolated polynucleotide obtained from an undefined library, preferably D NA (eg, cDNA) is determined under conditions that permit hybridization. Enables association with immobilized sequences from defined driver libraries.   Preferably, hybridization is under stringent conditions, for example, about 90% or more. Only identical sequences remain hybridized throughout the process Happens under such conditions. However, if desired, select other less stringent conditions. You may choose. For example, less stringent conditions promote hybridization To reduce the size of the subtraction library. Yo The initial hybridization was performed with very low stringency and Enables hybridization and allows for very small subtraction libraries Good. Subtraction lanes with increased or increased subsequent stringency You may control the size and content of the library.   Methods and conditions for hybridization at a selected stringency, For example, the conditions described herein are well known in the art. For example, Sa mbrook et al., MOLECULAR CLONING, A LABORARTORY MANUAL, 2nd Ed, Cold Spr See ing Harbor Laboratory Press, Cold Spring Harbor, NY 1989.   Multiple rounds, preferably using the conditions described elsewhere herein Preferably, hybridization is performed. Hybridization round Most preferably, the number is about 3 to 6. V.Isolation of nonhybridized DNA   After hybridization is complete, hybridize to the immobilized sequence on the grid surface. The undefined "undefined" sequence was hybridized using standard methods. Collect from purification solution and purify. See, for example, Sambrook et al., Supra.   In a preferred embodiment in which DNA is produced using the phagemid system, Hybridization is achieved by synthesizing the second strand of unhybridized DNA. The non-ionized DNA is converted to a double-stranded plasmid. Appropriate in appropriate cells The resulting plasmid is propagated under suitable conditions. Those skilled in the art will recognize the type of plasmid used. Considering the type, an appropriate host cell can be easily determined. However, desirable Selects host cells from bacterial cells. Currently, the preferred bacterial cell is E. coli ( E. coli) strain. After cell culture, the resulting colonies are transformed into D using conventional methods. Isolate NA.   In another embodiment, the use of a label sequence allows the library to be A sequence common to immobilized sequences derived from a collection to be Can be specified. In particular, according to the present invention, Tag sequences can be easily detected directly, which allows Identifying sequences that would be removed from the subtraction library it can. The sequences detected are in undefined libraries and defined An array common to column collections.   A collection of DNA isolated from a colony is a subtractive library of the present invention. To form This subtraction library contains libraries containing undefined arrays. , Which are characterized by containing DNA sequences present in the Excludes sequences that were in the collection of defined sequences fixed above . Methods for maintaining these subtraction libraries are well known to those skilled in the art.   The DNA in the obtained subtraction library was subjected to standard protocol [Sambro ok et al. or the ABI Prizm sequencing kit, Foster City, CA]. Or may be used for various other purposes. VI.Subtraction library   Therefore, the present invention provides a subtraction library produced by the method of the present invention. You. This subtraction library contains, inter alia, novel genes and gene fragments. Provides a source of novel nucleotide sequences that include These arrays, in particular Genes and gene fragments can be useful for screening drug candidates. The information obtained thereby can be used in the pharmaceutical industry to identify new drugs. You.   In addition, these sequences can be used in a conventional manner to isolate or An encoded peptide may be produced. Production of the protein or peptide of the present invention The polynucleotide of the desired gene of the present invention identified by using the method of the present invention. Inserting the nucleotide sequence, preferably DNA or a portion thereof, into a suitable expression system. You. In a preferred embodiment, a polynucleotide encoding a protein or peptide Otide sequence operably linked to heterologous expression control sequence allowing expression of human protein. Construct a recombinant molecule or vector that has been used. Mammals (including humans), Kon Many types of suitable expression vectors for insect, yeast, fungal and bacterial expression And host cell systems are known in the art.   In a suitable host cell, such as a mammal, bacterium, fungus or insect, or in a suitable host cell. Transfection of these vectors into irs yields the expressed protein. G Suitable host cells, cell lines and viruses for transfection and such Methods for construction and transfection of host cells and viruses are well known. I have. Transfection, culture, amplification, screening and product production And suitable methods for purification are also known in the art.   In one embodiment, the nucleotides identified according to the invention and thereby the The encoded protein or peptide is converted to a disease or susceptibility by conventional diagnostic assays. It can be used as a diagnostic composition useful for dye diagnosis. For example, animal creatures Diagnostic tests that detectably target the nucleotide sequence or protein of the present invention in biological samples Drugs can be developed. Such reagents include complementary nucleotide sequences, antibodies (mono- Clonal, recombinant or polyclonal), or chemically derived jaws Nist or A It may be an antagonist. Alternatively, the nucleotides of the present invention and the Encoded proteins or peptides, their fragments, or The capture sequence itself may be used as a diagnostic reagent. These reagents, for example, It may be labeled with a sex isotope or a colorimetric enzyme. Appropriate diagnostic assays The choice of format and detection system is within the skill of the artisan and requires further explanation. It can be easily selected without doing.   In addition, the genes and proteins or other sequences identified according to the invention may be used therapeutically. May be used. For example, a nucleic acid identified using the subtraction library of the present invention Otides, proteins or peptides can be used to convert natural or synthetic compounds useful as therapeutics. It may serve as a target for cleaning and development. Gene expression or Compounds that inhibit proteins are also considered therapeutically useful. In addition, genes essential for organisms May be used.   Conventional assays and methods may be used to screen for such agents. For example, it specifically binds or binds to the protein encoded by these nucleotide sequences. A simple method for identifying compounds that inhibits is to test selected proteins or gene products The test compound to the compound by contact with the compound and then to the protein. It may involve measuring the amount of the test compound (if present). Heel The method involves incubating the test compound and the protein immobilized on a solid support. May be included. Yet Other Conventional Drug Screening The method involves using a suitable computer program to generate the gene product or a portion thereof. Determine compounds that have a similar or complementary structure to the And screening these compounds. Same Compound alone or in combination with other active ingredients in a suitable therapeutic regimen Is also good. Methods for formulating therapeutic compositions and suitable pharmaceutical carriers are well known to those skilled in the art. ing.   Thus, through the use of such a method, the present invention allows these genes (or other Or the protein encoded by them or a part thereof Capable of interacting with and optionally enhancing or reducing biological activity It is thought to provide a compound. Thus, these compounds are also within the scope of the present invention. . VII.Subtraction probe   Furthermore, the present invention provides a probe obtained by subtraction according to the method of the present invention. I do. These methods can be performed using the method described below or the method described anywhere in this specification. May be obtained. A mixture of unknown polynucleotides, preferably DNA A subtraction probe may be created by subtracting a known gene from the gene. Book After a subtraction round according to the invention, the remaining polynucleotides are, for example, Prototype for probing unknown polynucleotides by hybridization methods Is the This can be accomplished using well-known methods, for example using colorimetric or radioactive labels. These probes may be labeled. These subtraction probes, among others, Providing a source of novel nucleotide sequences, including genes and gene fragments; They are known polynucleotide sequences, especially in mixtures and libraries. Particularly useful for detecting unknown polynucleotide sequences in solution or in solution It is. These probe sequences, and especially genes and gene fragments Are useful in screening target candidates for drug screening. Can be for Use the resulting information in the pharmaceutical industry to identify new drugs Can be   Initiate a polynucleotide amplification reaction, such as PCR, using a subtraction probe. You may. To do this, a priming site is provided at at least one end of the molecule. Subtraction probes can be created using degenerate polynucleotide sequences containing You. Two priming sites may be added to the molecule, one at each end. May be the same sequence or different sequences. For example, about 5 A priming site, which is a known sequence of 40 nucleotides, is ligated using ligase. It may be added to the subtraction probe. These subtraction probes can be used in solution or It may be used as a primer for amplification on a body support. The preferred way And these primers are used as a collection of polynucleotides on a solid support. Hybridize. After hybridization, amplification Or double-stranded polynucleotide such as double-stranded DNA by PCR be able to. Polynucleotide primers useful for amplification include a priming site An array that is complementary to one or more And more than one, they are different. For example known methods or Methods described herein, eg, a zero blunt end kit from In Vitrogen (Carlsbad, CA) May be used to clone the amplified fragment.   In addition, these probe sequences are used in a conventional manner to The genes may be obtained by means of the Isolated proteins or peptides may be produced. Anywhere in this document The protein or peptide of the present invention may be produced using the method described in (1).   In one embodiment, the probe nucleotide identified according to the present invention is Diseases by methods described elsewhere in the handbook or by methods known in the art Alternatively, it can be used as a diagnostic composition useful in diagnosing infection.   These examples illustrate a preferred method of the present invention. These examples are illustrative It is for injuries and does not limit the scope of the invention.Example 1-Production of a subtraction library A. Immobilization of a collection of known cDNA sequences   A driver collection containing defined DNA is shown on a solid glass table as shown below. Grid on the surface. The collection is transferred to a pBluescript vector (Stratagene, La J olla, CA), and cloned into plasmids by PCR using vector-specific primers. Collect the sheet. Deposit insert containing cDNA on glass surface by capillary And attach using standard methods [Silicon Compounds: Register and Review (Uni ted Chemical Technologies, Inc., Bristol, Pennsylvania, (1993)].B. Formation of an undefined cDNA library   An undefined cDNA library from the desired source can be substituted for the modified vector. The SuperScript system (Life Technologi es, Gaithersburg, MD). Briefly, the desired clonin PBluescript to remove the multiple cloning site In order to avoid hybridization, pUC118 [J. Vieira and Messing, Met hods in Enzymology, 153: 3 (1987)]. Deletion of undesired sequences Manufacturer's Quick Change Mutagenesis kit (Stratag) by protocol or another conventional method ene, La Jolla, CA). As bacteriophage, for example Enables isolation of single-stranded DNA packaged as a phagemid vector The vector is pre-processed to include the sequence.C. subtraction   Using helper phage according to the method described in Sambrook et al. Single-stranded DNA from the library to be calculated (cDNA library described in B) Isolate. One using 4 × SSC, 42 ° C., 16 to 18 hours, 20 μl Hybridization of the strand library with the gridded library described in A. Let it. After an appropriate hybridization time, the hybridization solution is Remove and precipitate using standard methods (Sambrook et al., Supra). In the preferred embodiment And multiple rounds of hybridization, preferably using the conditions of Example 1C. Perform Most preferably, the number of hybridization rounds is about 4.   Alternatively, hybridization may be performed at a lower temperature, for example, at 37 ° C. No. After hybridization, first collect the hybridization solution, Then, process as follows. The grid was washed with 2X SSC at 37 ° C. The washings are collected and then precipitated. DNA is recovered as described below. Then These steps are repeated at a higher temperature, for example at 65 ° C, and then at both temperatures. And wash with 0.2X SSC.   After hybridization, the precipitated DNA was subjected to standard procedures (Sambrook et al., Supra). To convert to double-stranded DNA using Ref. Briefly, vector-specific The oligonucleotide is hybridized to the library, Synthesis of the second strand by E. coli DNA polymerase in the presence of 4 ligases Complete the reaction.   The obtained double-stranded DNA is transformed into an appropriate E. coli host strain (for example, DH5alpha, Life Sci. ences Technology, Gaithersburg, MD) The obtained colonies are collected to obtain a subtraction library.   All publications and references cited herein, not limited to patents and patent applications. To Each is hereby incorporated by reference in its entirety. This application is excellent Patent applications claiming priorities are also incorporated by reference in their entirety, as are the above publications and literature. Are considered as described herein.   The above description fully discloses the invention including preferred embodiments of the invention. special Modifications and improvements of the embodiments disclosed herein are within the scope of the following claims. . Without further elaboration, the above description can be used to maximize the present invention. I am convinced. Therefore, the embodiments described herein are merely illustrative, It should be understood that they do not limit the scope of the invention in any way. Books for which exclusive rights have been claimed Examples of the invention are defined below.

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Claims (1)

[Claims]   1. The following steps:   (A) preparing a surface on which a collection containing a known nucleic acid sequence is immobilized,   (B) hybridizing a library of nucleic acid sequences containing undefined sequences to the surface Let it dise, then   (C) isolating non-hybridizing nucleic acids And a method for producing a nucleic acid subtraction library.   2. The method of claim 1, wherein the surface is glass.   3. The method according to claim 1, wherein the nucleic acid isolated from the library is DNA.   4. The method according to claim 3, wherein the DNA is single-stranded.   5. The method of claim 1, wherein the nucleic acids of the collection are DNA.   6. The method according to claim 5, wherein the DNA is cDNA.   7. The library according to claim 2, wherein the library is produced using a phagemid vector. Method.   8. The library according to claim 7, wherein the library is produced using the pUC118 vector. Method.   9. (A) synthesize the second strand of non-hybridized DNA Obtaining dimid DNA,   (B) growing the phagemid,   (C) isolating at least one clone Wherein the DNA that does not hybridize is isolated by the step comprising: Item 7. The method according to Item 7.   10. 10. The method of claim 9, wherein the phagemid is grown in a bacterial cell.   11. The method according to claim 2, wherein the library is produced using a phage vector. Law.   12. Active phage by synthesizing the second strand of DNA that does not hybridize A process that involves obtaining DNA, growing phage, and then isolating plaques. 12. The method according to claim 11, wherein the DNA that does not hybridize is isolated. Method.   13. 2. The method of claim 1, wherein the library is produced from a mixed set of RNA. .   14． 2. The method according to claim 1, wherein the library is a human cDNA library.   15. A subtraction library manufactured according to the method of claim 1.   16. 2. Isolated from a subtraction library produced according to the method of claim 1. Array.   17． An isolated protein produced by expression of the sequence of claim 15.   18. Library containing undefined array for existence of defined array And a method for rapidly screening cells, comprising:   (A) providing a surface having a collection containing known nucleic acid sequences;   (B) Representing nucleic acid sequences isolated from libraries containing undefined sequences Hybridization to the surface (the sequence is labeled) and then   (C) detecting the hybridized nucleic acid sequence A method that includes   19. 19. The method according to claim 18, wherein the nucleic acid sequence isolated from the library is DNA. Method.