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Gene expression analysis using array with immobilized tags of more than 25 bp (supersage-array)Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidGene expression analysis using array with immobilized tags of more than 25 bp (supersage-array) description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070172854, Gene expression analysis using array with immobilized tags of more than 25 bp (supersage-array). Brief Patent Description - Full Patent Description - Patent Application Claims
CLAIM PRIORITY [0001] The present application claims priority from Japanese applications JP2005-359366 filed on Dec. 13, 2005 and JP2006-138515 filed on May 18, 2006, the content of which is hereby incorporated by reference into this application. TECHNICAL FIELD [0002] The present invention relates to a method of gene expression analysis. More particularly, the present invention relates to a method of gene expression analysis that enables highly reproducible and high-throughput analysis, with the use of a microarray with immobilized improved SAGE.TM. tags of more than 25 bp. BACKGROUND ART [0003] Techniques for transcript analysis, such as microarray analysis and serial analysis of gene expression (SAGE.TM.), are indispensable for various types of biological research. Use of a microarray enables the expression analysis of large quantities of genes at one time and simultaneous analysis of multiple samples. With the use of a microarray, however, expression analysis can only be conducted exclusively for the genes spotted on the array. Accordingly, it is necessary to prepare an array upon which all relevant genes may be spotted, in order to perform extensive analysis. In the case of model organisms, such as rice or Arabidopsis thaliana, cDNA arrays or oligonucleotide arrays covering all genes thereof are commercially available and are generally employed in research. Concerning many other organisms, however, researchers are required to independently design arrays from cDNA libraries. This requires large amounts of time and cost. [0004] In contrast, serial analysis of gene expression (SAGE.TM.) enables the search for novel genes and the quantitative expression analysis thereof (Velculescu et al., Science 270: 484-487, 1995). With this technique, the genes are identified based on a 10- or 11-bp sequence located downstream of the restriction enzyme site (CATG), which is located closest to the 3'-end of the transcript, and the expression levels of such genes are analyzed. Accordingly, sequential reading of the sequences located around the 3'-end with the use of a DNA sequencer enables the extensive expression analysis of genes, including unknown genes. However, SAGE.TM. is not substantially suitable for simultaneous analysis of multiple samples due to the large number of experimental steps required. In addition, 14-bp SAGE.TM. tags and 21-bp tags that are employed in LongSAGE.TM. (Saha et al., Nature Biotechnology 20, 508-512, 2002) are too short to assuredly identify genes. Thus, applications of such tags are restricted to model organisms. [0005] In recent years, the present inventors had developed the SuperSAGE system, which is an improvement over SAGE.TM. (WO 2004/099445; Gene expression analysis of plant host-pathogen interactions by SuperSAGE, Matsumura, H., Reich, S., Ito, A., Saitoh, H., Kamoun, S., Winter, P., Kahl, G., Reuter, M., Krueger, D., and Terauchi R., 2003, Proc. Natl. Acad. Sci. U.S.A., 100: 15718-15723; Molekulares Wechselspiel von Wirt und Pathogen: Simultane, genomweite Transkriptprofilierung zweier Organismen mit SuperSAGE, Kahl, G., Winter, P., Matsumura, H., Reuter, M., Kruger, D. and Terauchi R., 2004, Biospektrum 10: 511-513; SuperSAGE, Matsumura, H., Ito, A., Saitoh, H., Winter, P., Kahl, G., Reuter, M., Krueger, D. H. and Terauchi, R., 2005, Cellular Microbiology, 2005, 7: 11-18; and SuperSAGE, a potent transcriptome tool for eukaryotic Organisms, Matsumura, H., Reich, S., Reuter, M., Krueger, D. H., Winter, P., Kahl, G. and Terauchi R., In: S.-M. WANG (ed.) SAGE: Current Technologies and Applications, Horizon Scientific Press, 2004, 77-90). SuperSAGE involves the use of a type III restriction enzyme, EcoP15I, to obtain a 26-bp nucleotide sequence tag. Use of tags each of 26 bp remarkably improves the accuracy of gene identification. Such tags also enable simultaneous analysis of gene expression both in host cells and in pathogen cells, and applications thereof became available with regard to non-model organisms, for which no DNA database is available. DISCLOSURE OF THE INVENTION [0006] It is an object of the present invention to provide a method of gene expression analysis that enables extensive gene expression analysis and simultaneous analysis of multiple samples of organisms for which the genomic analysis has not yet been advanced. [0007] In order to attain the above object, the present inventors had examined whether or not tags each of 26 bp of SuperSAGE (SuperSAGE tags) could be utilized as probes for microarrays. As a result, they discovered that the results of gene expression analysis attained with the use of an array with immobilized SuperSAGE tags would be similar to those attained via conventional SAGE.TM., and that such results could be attained through a single hybridization step. Further, they also discovered that immobilization of SuperSAGE tags would produce unexpected effects, i.e., preparation of microarrays would be remarkably facilitated in non-model organisms for which no EST, cDNA, or genomic sequences are available. [0008] More specifically, the present invention relates to a solid support onto which tags each comprising an oligonucleotide of more than 25 bp for identifying the expressed genes are immobilized, wherein the 3'-end of the tag is defined by a cleavage site of a type III restriction enzyme and the 5'-end thereof is defined by a cleavage site of the other restriction enzyme located closest to the 3'-end of the cDNA of such genes. The present invention also relates to a method of gene expression analysis involving the use of such solid support with immobilized tags. [0009] The sequences of the tags according to the present invention (i.e., SuperSAGE tags) can be determined based on the SuperSAGE system that the present inventors previously developed (WO 2004/099445). Specifically, such tag sequences are determined in accordance with the following steps: [0010] 1) a cDNA pool is synthesized from mRNAs of expressed genes using a primer comprising a recognition sequence of a type III restriction enzyme and an oligo-dT sequence, and treating the cDNA pool with another restriction enzyme; [0011] 2) a poly(A)-containing fragment is purified from the cDNA pool, and such fragment is ligated to a linker A or B; [0012] 3) the fragment is treated with a type III restriction enzyme, and the resulting linker A-containing fragment is ligated to a linker B-containing fragment; [0013] 4) linker sequences are removed by cleaving the ligated fragments with another restriction enzyme used in step 1) to obtain ditag oligonucleotides; [0014] 5) ditag oligonucleotides are ligated to each other to prepare polynucleotides; and [0015] 6) the nucleotide sequences of the above polynucleotides are analyzed to determine the nucleotide sequences of tags contained in such polynucleotides. [0016] Examples of type III restriction enzymes that can be used in the present invention are disclosed at a web site (http://rebase.neb.com/cgi-bin/azlist?re3), and examples thereof include EcoPI and EcoP15I. [0017] Examples of other restriction enzymes (commercial products) include those shown in the table below. TABLE-US-00001 Recognition sequence Enzymes (commercial products only) CATG{circumflex over ( )} NlaIII, Hsp92II, {circumflex over ( )}CATG FatI C{circumflex over ( )}TAG BfaI, MaeI, XspI A{circumflex over ( )}CGT HpyCH4IV, MaeII, ACGT{circumflex over ( )} TaiI, TscI AG{circumflex over ( )}CT AluI T{circumflex over ( )}CGA TaqI {circumflex over ( )}GATC BfuCI, Bsp143I, BstENII, DpnII, Kzo9I, MboI, NdeII, Sau3AI GAT{circumflex over ( )}C BstKTI, G{circumflex over ( )}TAC Csp6I [0018] As a preferred embodiment of the present invention, use of EcoP15I and N1aIII is described in the Examples below. When EcoP15I and N1aIII are used, the aforementioned linker A and linker B are double-stranded DNAs that are different from each other and that are obtained by annealing the following first strand of DNA (1) and the second strand of DNA (2): TABLE-US-00002 DNA (1): 5'-N.sub.30-40-CAGCAGCATG-3' DNA (2): 3'-N.sub.30-40-GTCGTC-5' [0019] wherein "N.sub.30-40" of DNA (1) is complementary to "N.sub.30-40" of DNA (2), each thereof is a sequence comprising 30 to 40 arbitrary nucleotides, the 5'-end of DNA (1) may be labeled, and the 3'-end of DNA (2) may be amino-modified. [0020] The array according to the present invention may be prepared by synthesizing tag oligonucleotides on the solid support. Alternatively, the array may be prepared by immobilizing pre-synthesized tag oligonucleotides on the solid support. Continue reading about Gene expression analysis using array with immobilized tags of more than 25 bp (supersage-array)... 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