Gene expression analysis using array with immobilized tags of more than 25 bp (SuperSAGE-Array)

Abstract

This invention provides a method of gene expression analysis that enables extensive gene expression analysis and simultaneous analysis of multiple samples of organisms for which genomic analysis has not yet been advanced. In this method, tags each comprising an oligonucleotide of more than 25 bp for identifying expressed genes, 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 another restriction enzyme located closest to the 3′-end of the cDNA of such genes, are immobilized on a solid support, gene-containing samples are hybridized to the solid support, and the signals emitted from the genes hybridized to the tags are detected to analyze the gene expression profiles in the samples.

1 . A method of gene expression analysis, wherein tags each comprising an oligonucleotide of more than 25 bp for identifying expressed genes, 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 another restriction enzyme located closest to the 3′-end of the cDNA of the genes, are immobilized on a solid support, gene-containing samples are hybridized to the solid support, and the signals emitted from the genes hybridized to the tags are detected to analyze the gene expression profiles in the samples.  
     
     
         2 . The method according to  claim 1 , wherein the tags comprise nucleotide sequences determined by the following steps: 
 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;    2) a poly(A)-containing fragment is purified from the cDNA pool, and the fragment is ligated to a linker A or B;    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;    4) linker sequences are removed by cleaving the ligated fragments with another restriction enzyme used in step 1) to obtain ditag oligonucleotides;    5) ditag oligonucleotides are ligated to each other to prepare polynucleotides; and    6) the nucleotide sequences of the above polynucleotides are analyzed to determine the nucleotide sequences of tags contained in the polynucleotides.    
     
     
         3 . The method according to  claim 1 , wherein the type III restriction enzyme is EcoP15I.  
     
     
         4 . The method according to  claim 3 , wherein the another restriction enzyme is any of N1aIII, Hsp92II, FatI, BfaI, MaeI, XspI, HpyCH4IV, MaeII, TaiI, TscI, AluI, TaqI, BfuCI, Bsp143I, BstENII, DpnII, Kzo9I, MboI, NdeII, Sau3AI, BstKTI, or Csp6I.  
     
     
         5 . The method according to  claim 4 , wherein linker A and linker B are double-stranded DNAs different from each other and are obtained by annealing the following first strand of DNA (1) and second strand of DNA (2)  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   DNA (1): 
                     
                     
                 
                     
                   5′-N 30-40 -CAGCAGCATG-3′ 
                   (SEQ ID NO: 201) 
                 
                     
                     
                 
                     
                   DNA (2): 
                 
                     
                   3′-N 30-40 -GTCGTC-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
         wherein “N 30-40 ” of DNA (1) is complementary to “N 30-40 ” of DNA (2) and 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.  
       
     
     
         6 . A solid support on which tags each comprising an oligonucleotide of more than 25 bp for identifying expressed genes are immobilized, wherein the 3′-end of the tag is defined by a cleavage site of EcoP15I and the 5′-end thereof is defined by a cleavage site of N1aIII located closest to the 3′-end of the cDNA of the genes, and 
 wherein the tags comprises nucleotide sequences determined by the following steps:    1) a cDNA pool is synthesized from mRNAs of expressed genes using a primer comprising a recognition sequence of EcoP15I and an oligo-dT sequence, and treating the cDNA pool with N1aIII;    2) a poly(A)-containing fragment is purified from the cDNA pool, and such fragment is ligated to a linker A or B;    3) the fragment is treated with EcoP15I, and the resulting linker A-containing fragment is ligated to the linker B-containing fragment;    4) linker sequences are removed by cleaving the ligated fragments with N1aIII used in step 1) to obtain ditag oligonucleotides;    5) the ditag oligonucleotides are ligated to each other to prepare polynucleotides; and    6) the nucleotide sequences of the above polynucleotides are analyzed to determine the nucleotide sequences of tags contained in such polynucleotides.    
     
     
         7 . The solid support according to  claim 6 , wherein linker A and linker B are double-stranded DNAs different from each other that are obtained by annealing the following first strand of DNA (1) and second strand of DNA (2):  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   DNA (1): 
                     
                     
                 
                     
                   5′-N 30-40 -CAGCAGCATG-3′ 
                   (SEQ ID NO: 201) 
                 
                     
                     
                 
                     
                   DNA (2): 
                 
                     
                   3′-N 30-40 -GTCGTC-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
         wherein “N 30-40 ” of DNA (1) is complementary to “N 30-40 ” of DNA (2) and 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.  
       
     
     
         8 . The solid support according to  claim 6 , which is prepared by synthesizing tag oligonucleotides thereon.  
     
     
         9 . The solid support according to  claim 6 , which is prepared by immobilizing pre-synthesized tag oligonucleotides thereon.