| 主权项 |
1. A method of identifying a plurality of bases in a target nucleic acid, comprising the following steps:
(i) attaching the 5′-end of a primer to a solid surface; (ii) hybridizing a target nucleic acid to the primer attached to the solid surface; (iii) adding a compound of the formula: wherein:
Z is —O—, —S—, —NH—, —OC(O)O—, —NHC(O)O—, —OC(O)NH— or —NHC(O)NH—;R1 is triphosphate or α-thiotriphosphate;R2 is hydrogen or hydroxy;R3 and R4 are each independently:
hydrogen, hydroxy, halo or amino; oralkyl(C≦12), alkenyl(C≦12), alkynyl(C≦12), aryl(C≦12), aralkyl(C≦12), heteroaryl(C≦12), heteroaralkyl(C≦12), alkoxy(C≦12), aryloxy(C≦12), aralkoxy(C≦12), hetero-aryloxy(C≦12), heteroaralkoxy(C≦12), alkylamino(C≦12), dialkylamino(C≦12), arylamino(C≦12), aralkyl-amino(C≦12), or a substituted version of any of these groups; orR5, R7, and R8 are each independently:
hydrogen, hydroxy, halo, amino, nitro, cyano or mercapto;alkyl(C≦12), alkenyl(C≦12), alkynyl(C≦12), aryl(C≦12), aralkyl(C≦12), heteroaryl(C≦12), heteroaralkyl(C≦12), acyl(C≦12), alkoxy(C≦12), alkenyloxy(C≦12), alkynyl-oxy(C≦12), aryloxy(C≦12), aralkoxy(C≦12), heteroaryl-oxy(C≦12), heteroaralkoxy(C≦12), acyloxy(C≦12), alkylamino(C≦12), dialkylamino(C≦12), alkoxy-amino(C≦12), alkenylamino(C≦12), alkynylamino(C≦12), arylamino(C≦12), aralkylamino(C≦12), heteroaryl-amino(C≦12), heteroaralkylamino(C≦12), alkyl-sulphonylamino(C≦12), amido(C≦12), alkylthio(C≦12), alkenylthio(C≦12), alkynylthio(C≦12), arylthio(C≦12), aralkylthio(C≦12), heteroarylthio(C≦12), heteroaralkyl-thio(C≦12), acylthio(C≦12), thioacyl(C≦12), alkyl-sulfonyl(C≦12), arylsulfonyl(C≦12), alkyl-ammonium(C≦12), alkylsulfonium(C≦12), alkyl-silyl(C≦12), or a substituted version of any of these groups;R6 is a -linker-reporter;or a salt thereof; (iv) adding a nucleic acid replicating enzyme to the hybridized primer/target nucleic acid complex to incorporate the compound of step (iii) into the growing primer strand, wherein the incorporated compound of step (iii) terminates the polymerase reaction at an efficiency of between about 70% to about 100%; (v) washing the solid surface to remove unincorporated components; (vi) detecting the reporter group of the incorporated compound of step (iii); (vii) a photochemical cleavage step to remove the terminating moiety of the incorporated compound, wherein the terminating moiety has the formula: (viii) washing the solid surface to remove the cleaved terminating moiety; and (ix) repeating steps (iii) through (viii) one or more times to identify the plurality of bases in the target nucleic acid. |
