Method for gene identification signature (GIS) analysis

Abstract

An isolated oligonucleotide comprising at least one ditag, wherein the ditag comprises two joined first and second sequence tags, wherein the first tag comprises the 5′-terminus sequence and the second tag comprises the 3′-terminus sequence of a nucleic acid molecule or a fragment thereof. The ditag analysis is useful for gene discovery and genome mapping.

1 . An isolated oligonucleotide comprising at least one ditag, wherein the ditag includes two joined first and second sequence tags, and wherein the first tag includes a 5′-terminus sequence and a second tag comprises the 3′-terminus sequence of a nucleic acid molecule or fragment thereof.  
     
     
         2 . The oligonucleotide of  claim 1 , further comprising two adapters flanking the ditag, wherein each adapter includes at least one restriction site.  
     
     
         3 . The oligonucleotide of  claim 2 , wherein each adapter comprises at least a first restriction site which is an asymmetric restriction site and at least a second restriction site.  
     
     
         4 . The oligonucleotide of  claim 3 , wherein the asymmetric restriction site comprises a type II restriction site.  
     
     
         5 . The oligonucleotide of  claim 1 , wherein the nucleic acid molecule comprises the full-length sequence of a gene or a fragment thereof.  
     
     
         6 . The oligonucleotide of  claim 1 , wherein the nucleic acid comprises RNA, mRNA, genomic DNA, full-length cDNA, or cDNA.  
     
     
         7 . The oligonucleotide of  claim 1 , wherein the ditag is obtained by splicing the 5′ terminus and the 3′ terminus of the nucleic acid molecule or fragment thereof in the presence of at least one restriction enzyme and the size of the sequence tags is determined by the restriction enzyme used.  
     
     
         8 . The oligonucleotide of  claim 7 , wherein the restriction enzyme is a type TI restriction enzyme.  
     
     
         9 . The oligonucleotide of  claim 7 , wherein the restriction enzyme is selected from the group consisting of AarI, AceIII, AloI, BaeI, Bbr7I, BbvI, BbvII, BccI, Bce83I, BceAI, BcefI, BcgI, BciVI, BfiI, BinI, BplI, BsaXI, BscAI, BseMII, BseRI, BsgI, BsmI, BsmAI, BsmFI, Bsp24I, BspCNI, BspMI, BsrI, BsrDI, BstF51, BtgZI, BtsI, CjeI, CjePT, EciI, Eco31I, Eco57I, Eco57MI, Esp3I, FalI, FauI, FokI, GsuI, HaeIV, HgaI, Hin4I, HphI, HpyAV, Ksp632I, MboII, MlyI, MmeI, MnlI, PleI, PpiI, PsrI, RleAI, SapI, SfaNI, SspD51, Sth132I, StsI, TaqII, TspDTI, TspGWI, TspRI, Tth111II, I-CeuI, PI-SceI, PI-PspI and I-SceI.  
     
     
         10 . The oligonucleotide of  claim 1 , wherein the ditag size is 12-60 bp.  
     
     
         11 . The oligonucleotide of  claim 1 , wherein the ditag comprises 34-38 nucleotides and the size of each tag is determined by the use of restriction enzyme Mmel.  
     
     
         12 . The oligonucleotide of  claim 1 , wherein the first and second tag have the same or a different number of nucleotides.  
     
     
         13 . The oligonucleotide of  claim 1 , wherein the oligonucleotide consists of 1 to 1000 ditags.  
     
     
         14 . A vector comprising an isolated oligonucleotide including at least one ditag, wherein the ditag includes two joined first and second sequence tags, and wherein the first tag includes a 5′-terminus sequence and a second tag comprises the 3′-terminus sequence of a nucleic acid molecule or fragment thereof.  
     
     
         15 . The vector of  claim 14  further comprising two adapters flanking the ditag, wherein each adapter includes at least one restriction site and wherein each adapter includes at least a first restriction site which is an asymmetric restriction site and at least a second restriction site.  
     
     
         16 . The vector of  claim 15 , wherein the backbone of the vector does not comprise the asymmetric restriction site or the second restriction site.  
     
     
         17 . The vector of  claim 16 , wherein the asymmetric restriction site is a type II restriction site.  
     
     
         18 . A vector comprising at least a nucleic acid molecule and two adapters flanking the nucleic acid molecule, wherein each adapter comprises at least: a first restriction site which is a type II restriction site and at least a second restriction site, and wherein the backbone of the vector does not comprise the type II restriction site, or the second restriction site.  
     
     
         19 . The vector of  claim 18 , wherein the type T1 restriction site is MmeI.  
     
     
         20 . A vector comprising SEQ ID NO:18.  
     
     
         21 . The vector of  claim 20 , comprising at least one ditag, wherein the ditag includes two joined first and second sequence tags, and wherein the first tag includes a 5′-terminus sequence and a second tag comprises the 3′-terminus sequence of a nucleic acid molecule or fragment thereof.  
     
     
         22 . The vector of  claim 21 , comprising comprising two adapters flanking the ditag, wherein each adapter includes at least one restriction site and wherein each adapter includes at least a first restriction site which is an asymmetric restriction site and at least a second restriction site.  
     
     
         23 . A cDNA library, wherein every cDNA clone of the library comprises at least one oligonucleotide including at least one ditag, wherein the ditag includes two joined first and second sequence tags, and wherein the first tag includes a 5′-terminus sequence and a second tag comprises the 3′-terminus sequence of a nucleic acid molecule or fragment thereof.  
     
     
         24 . The cDNA library of  claim 23 , wherein the at least one oligonucleotide comprises 1-1000 ditags.  
     
     
         25 . A method for recovering full-length cDNA comprising: 
 preparing, from a full-length cDNA library, at least one oligonucleotide including at least one ditag, the ditag including two joined first and second sequence tags, wherein the first tag includes the 5′-terminus sequence and the second tag includes the 3′-terminus sequence of a full-length cDNA;    sequencing the obtained oligonucleotide ditag;    determining the ditag of interest; and    recovering the full-length cDNA corresponding to the ditag of interest from the full-length cDNA library.    
     
     
         26 . A method for quantifying the transcriptional activity of a gene comprising: 
 preparing, from a full-length cDNA library, at least one oligonucleotide comprising at least one ditag, the ditag including two joined first and second sequence tags, wherein the first tag includes the 5′-terminus sequence and the second tag includes the 3′-terminus sequence of a full-length cDNA;    sequencing the obtained oligonucleotide ditag;    determining the frequency of the sequenced ditag which corresponds to the transcriptional activity of the gene.