METHOD TO GENERATE OR DETERMINE NUCLEIC ACID TAGS CORRESPONDING TO THE TERMINAL ENDS OF DNA MOLECULES USING SEQUENCES ANALYSIS OF GENE EXPRESSION (TERMINAL SAGE)

摘要

92 ABSTRACT1IET'HOD TO CliiNNERr9T'E OtR D TERMITNE NUCLEIC ACID C.5 CORRESPONDING TO THE TERMINAL ENDS OF TINA MO EC Its USING SEQUENCES ANALYSIS OF GENE EXPRESSION (TER-1TT AL SAGE"We describe a method of wgeneraling a database comprisir a plurality of records, each record comprising all indication whether a genie is expres by a particular cell; the rnethod comprising ^,wls of (a) providing a first nut.€eic. r d sequence; (h) linking tire. first nucleic acid sequence to a taker sequence to On linked nucleic acid, in which VAthe linker sequence comprises: (i) a first recognition for a first nucleic acid cleavage enzyme that allows cleavage of the first nuclei h sequence at a site distant from the first recognition sue, and (n) a second rtcogniti' Fitt fir a second nucleic acid leaIare enzyme that allows: nucleic acid Cleavage at she distant from the Second recognition ite saR cle'aal e she located at a poShot whim or about the first recognition site in Much the linked nucleic acid has ih SfrlistFlre. 5' --- second recognition site -- first recognition site -• first nucleic acid (c) ele lvinrt the, linked nucleic acid with the first nucleic acid cleavage enzyme to twice (0 a linked tag comprising the linker sequence linked to a rlfu.I tide alulan t1g !' resent tine of the first nucleic acid. sequence and comprising a tel'hrliFhi.ll l?Oit1. tleere'.ioi: and (11) a second nucleic acid sequence conlprisiing a remainder par ion of e first nu heir acid; (d) deteetiSl die preseI ce, S quence oretude sequence tag to provide. an indieation (if an •~ -y of the linked or the nul _ identityinst;ince of gene •` 'resshon; and honing an indicluon of epsessien of the gene as a record in the da' use.'iguine:3ABSTRACTMETHOD OF GENERATING A DATABASEWe describe 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 an linker sequence fu 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 she distant from the first recognition site, and (ii) a second recognition site for a second nucleic acid cleavage sites said cleavage site located at a position wilthin or about 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 finked tag, comprising the linker sequence linked to a nuleotide 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 fist nucleic acid; (d) detecting the presence, sequence or identity of the lurked 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,Figure 3