Method to generate or determine nucleic acid tags corresponding to the terminal ends of dna molecules using sequences analysis of gene expression (terminal sage)

Abstract

We describe a method of providing an indication of an instance of expression of a gene, the method comprising the steps of: (a) providing a complementary deoxyribonucleic acid (cDNA) having a terminus comprising a terminal transcribed sequence of a gene; (b) linking the cDNA to an linker sequence thereby forming a linked nucleic acid, in which the linker sequence comprises a first recognition site for a first nucleic acid cleavage enzyme, preferably a restriction endonuclease, that allows nucleic acid cleavage at a site distant from the first recognition site; and (c) cleaving the linked nucleic acid with the first nucleic acid cleavage enzyme to provide a linked tag, in which the linked tag comprises a nucleotide sequence tag representative of a terminal transcribed sequence of the gene; and (d) detecting the presence or identity of the linked tag or the nucleotide sequence tag to provide an indication of an instance of gene expression.

1 . A method of generating a database comprising a plurality of records, each record comprising an indication whether a gene is expressed by a particular cell, the method comprising steps of:
 (a) providing a first nucleic acid sequence;   (b) linking the first nucleic acid sequence to a linker sequence to form a linked nucleic acid, in which the linker sequence comprises:
 (i) a first recognition site for a first nucleic acid cleavage enzyme that allows cleavage of the first nucleic acid sequence at a site distant from the first recognition site, and 
 (ii) a second recognition site for a second nucleic acid cleavage enzyme that allows nucleic acid cleavage at a site distant from the second recognition site, said cleavage site located at a position within the first recognition site; 
   in which the linked nucleic acid has the structure: 5′—second recognition site—first recognition site—first nucleic acid—3′   (c) cleaving the linked nucleic acid with the first nucleic acid cleavage enzyme to provide:
 (i) a linked tag comprising the linker sequence linked to a nucleotide sequence tag representative of the first nucleic acid sequence and comprising a terminal portion thereof; and 
 (ii) a second nucleic acid sequence comprising a remainder portion of the first nucleic acid. 
   (d) detecting the presence, sequence or identity of the linked tag or the nucleotide sequence tag to provide an indication of an instance of gene expression; and   
       storing an indication of expression of the gene as a record in the database. 
     
     
         2 . The database generation method of  claim 1 , in which the first nucleic acid comprises a complementary deoxyribonucleic acid (cDNA) having a terminus comprising a 5′ terminal transcribed sequence of a gene, and in which the linker sequence is ligated to said terminus. 
     
     
         3 . The database generation method of  claim 1 , in which the first nucleic acid comprises a complementary deoxyribonucleic acid (cDNA) having a terminus comprising a 3′ terminal transcribed sequence of a gene, and in which the linker sequence is ligated to said terminus. 
     
     
         4 . The database generation method of  claim 1 , in which the second nucleic acid cleavage enzyme recognition site comprises a MmeI restriction endonuclease cleavage site. 
     
     
         5 . The database generation method of  claim 1 , in which the first nucleic acid cleavage enzyme recognition site comprises restriction endonuclease BseRI cleavage site. 
     
     
         6 . The database generation method of  claim 1 , in which the method comprises sequentially generating a plurality of nucleic acid sequences each comprising a nucleotide sequence tag from a nucleic acid, the method comprising repeating steps (a) to (c) of  claim 1  at least once, in which the first nucleic acid sequence of step (a) is provided by the second nucleic acid sequence of step (c)(ii); and storing each indication of an instance of gene expression generated as a record in the database. 
     
     
         7 . A method of generating a database comprising a plurality of records, each record comprising an indication whether a gene is expressed by a particular cell, the method comprising the steps of:
 (a) providing a first linked tag and a second linked tag, each independently produced by the method of  claim 1 ;   (b) linking the first linked tag and the second linked tag such that the nucleotide sequence tag portion of one linked tag is linked to the nucleotide sequence tag of the other linked tag to form a ditag, the ditag comprising terminal transcribed sequences from first and second genes; and   (c) detecting the presence or identity of the ditag, or at least one nucleotide sequence tag comprised therein, to detect gene expression; and   
       storing the indication of expression of the gene as a record in the database. 
     
     
         8 . The database generation method of  claim 7 , further comprising the steps of:
 (d) cleaving the ditag with the second nucleic acid cleavage enzyme;   (e) linking a plurality of the resultant trimmed ditags to form a concatamer; and   (f) obtaining the nucleic acid sequence of at least a portion of the concatamer.   
     
     
         9 . A method of generating a database comprising a plurality of records, each record comprising an indication whether a gene is expressed by a particular cell, the method comprising:
 (a) obtaining an indication of an instance of expression of a gene by a method comprising:
 (i) providing a complementary deoxyribonucleic acid (cDNA) having a terminus comprising a terminal transcribed sequence of a gene; 
 (ii) linking the cDNA to a linker sequence, thereby forming a linked nucleic acid, in which the linker sequence comprises a first recognition site for a first nucleic acid cleavage enzyme that allows nucleic acid cleavage at a site distant from the first recognition site; and 
 (iii) cleaving the linked nucleic acid with the first nucleic acid cleavage enzyme to provide a linked tag, in which the linked tag comprises a nucleotide sequence tag representative of a terminal transcribed sequence of the gene; and 
 (iv) detecting the presence or identity of the linked tag or the nucleotide sequence tag to provide an indication of an instance of gene expression; and 
   
       storing the indication of expression of the gene as a record in the database. 
     
     
         10 . The database generation method according to  claim 9 , in which the cDNA comprises the 5′ terminal transcribed sequence of the gene, or the 3′ terminal transcribed sequence of the gene, or both. 
     
     
         11 . The database generation method according to  claim 9 , in which the cDNA is full length cDNA, comprising substantially all the coding sequence of the gene. 
     
     
         12 . The database generation method according to  claim 9 , in which the linker sequence further comprises a second recognition site for a second nucleic acid cleavage enzyme, that allows nucleic acid cleavage at a site distant from the second recognition site, and in which the second recognition site is located 5′ of the first recognition site in the linker sequence. 
     
     
         13 . The database generation method according to  claim 9 , in which the first nucleic acid cleavage enzyme recognition site comprises a Mme1 recognition site 5′-TCC GAC-3′. 
     
     
         14 . The database generation method according to  claim 12 , in which the second nucleic acid cleavage enzyme recognition site comprises a BseRI recognition site 5′-GAGGAG-3′. 
     
     
         15 . The database generation method of  claim 1 , further comprising storing the database in a computer readable medium. 
     
     
         16 . A method of generating a computer readable medium comprising a database comprising a plurality of records which indicate whether a gene is expressed by a particular cell, the method comprising generating a database by a method of  claim 1  and storing the database on the computer readable medium. 
     
     
         17 . A computer readable medium comprising a database generated according to the method of  claim 1 . 
     
     
         18 . A method of generating a database comprising a plurality of records, each record comprising an indication whether a gene is expressed by a particular cell, the method comprising steps of:
 (a) providing a first nucleic acid sequence comprising a full-length first strand cDNA comprising a 5′ untranslated region, a coding sequence, a 3′ untranslated region and a poly A tail;   (b) without prior cleavage of said first nucleic acid sequence, linking the 5′ end of said full length first strand cDNA to a linker sequence to form a linked nucleic acid, in which the linker sequence comprises:
 (i) a first recognition site for a first nucleic acid cleavage enzyme that allows cleavage of the first nucleic acid sequence at a site distant from the first recognition site, and 
 (ii) a second recognition site for a second nucleic acid cleavage enzyme that allows nucleic acid cleavage at a site distant from the second recognition site, said cleavage site located at a position within the first recognition site; in which 
   the linked nucleic acid has the structure: 5′—second recognition site—first recognition site—first nucleic acid—3′   (c) cleaving the linked nucleic acid with the first nucleic acid cleavage enzyme to provide:
 (i) a linked tag comprising the linker sequence linked to a nucleotide sequence tag representative of the first nucleic acid sequence, the nucleotide sequence tag comprising the 5′ terminus of the full length first strand cDNA; and 
 (ii) a second nucleic acid sequence comprising a remainder portion of the first nucleic acid; 
   (d) detecting the presence, sequence or identity of the linked tag or the nucleotide sequence tag to provide an indication of an instance of gene expression; and   storing an indication of expression of the gene as a record in the database.   
     
     
         19 . A system for generating a database comprising a plurality of records, each record comprising an indication whether a gene is expressed by a particular cell, the system comprising a programmable computer comprising a processor, a data storage system, at least one input device, and at least one output device, wherein the database is generated by the steps of:
 (a) providing a first nucleic acid sequence comprising a full-length first strand cDNA comprising a 5′ untranslated region, a coding sequence, a 3′ untranslated region and a poly A tail;   (b) without prior cleavage of said first nucleic acid sequence, linking the 5′ end of said full length first strand cDNA to a linker sequence to form a linked nucleic acid, in which the linker sequence comprises:
 (i) a first recognition site for a first nucleic acid cleavage enzyme that allows cleavage of the first nucleic acid sequence at a site distant from the first recognition site, and 
 (ii) a second recognition site for a second nucleic acid cleavage enzyme that allows nucleic acid cleavage at a site distant from the second recognition site, said cleavage site located at a position within the first recognition site; in which 
   the linked nucleic acid has the structure: 5′—second recognition site—first recognition site—first nucleic acid—3′   (c) cleaving the linked nucleic acid with the first nucleic acid cleavage enzyme to provide:
 (i) a linked tag comprising the linker sequence linked to a nucleotide sequence tag representative of the first nucleic acid sequence, the nucleotide sequence tag comprising the 5′ terminus of the full length first strand cDNA; and 
 (ii) a second nucleic acid sequence comprising a remainder portion of the first nucleic acid; 
   (d) detecting the presence, sequence or identity of the linked tag or the nucleotide sequence tag to provide an indication of an instance of gene expression; and   (e) storing an indication of expression of the gene as a record in the database.   
     
     
         20 . A tag code database access system, the system comprising a programmable computer comprising a processor, at least one input device, and at least one output device and a data storage system, the data storage system comprising a database of nucleic acid sequence tag codes generated by a method comprising:
 (a) providing a first nucleic acid sequence comprising a full-length first strand cDNA comprising a 5′ untranslated region, a coding sequence, a 3′ untranslated region and a poly A tail;   (b) without prior cleavage of said first nucleic acid sequence, linking the 5′ end of said full length first strand cDNA to a linker sequence to form a linked nucleic acid, in which the linker sequence comprises:
 (i) a first recognition site for a first nucleic acid cleavage enzyme that allows cleavage of the first nucleic acid sequence at a site distant from the first recognition site, and 
 (ii) a second recognition site for a second nucleic acid cleavage enzyme that allows nucleic acid cleavage at a site distant from the second recognition site, said cleavage site located at a position within the first recognition site; in which 
   the linked nucleic acid has the structure: 5′—second recognition site—first recognition site—first nucleic acid—3′   (c) cleaving the linked nucleic acid with the first nucleic acid cleavage enzyme to provide:
 (i) a linked tag comprising the linker sequence linked to a nucleotide sequence tag representative of the first nucleic acid sequence, the nucleotide sequence tag comprising the 5′ terminus of the full length first strand cDNA; and 
 (ii) a second nucleic acid sequence comprising a remainder portion of the first nucleic acid; 
   (d) detecting the presence, sequence or identity of the linked tag or the nucleotide sequence tag to provide an indication of an instance of gene expression; and   (e) storing an indication of expression of the gene as a record in the database.