| 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 to form a hybridized primer/target nucleic acid complex; (iii) obtaining a polymerase and a compound of formulas I-VII, with the proviso that compounds of different formulas I-VII have different reporters: wherein:
R1 is triphosphate or α-thiotriphosphate;R2 is hydrogen or hydroxy;R3 is alkyl(C≦8) or substituted alkyl(C≦8);R4 is
hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;alkyl(C≦6), acyl(C≦6), alkoxy(C≦6), acyloxy(C≦6), alkylamino(C≦6), dialkylamino(C≦6), amido(C≦6), or a substituted version of any of these groups;R5 is a -linker-reporter, wherein the reporter is based on a dye;R6 is:
hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;alkyl(C≦6), alkenyl(C≦6), alkynyl(C≦6), aryl(C≦6), aralkyl(C≦8), heteroaryl(C≦6), acyl(C≦6), alkoxy(C≦6), acyloxy(C≦6), alkylamino(C≦6), dialkylamino(C≦6), amido(C≦6), or a substituted version of any of these groups;or a salt, tautomer, or optical isomer thereof; (iv) reacting the hybridized primer/target nucleic acid complex with a polymerase and one or more of the compounds of step (iii) to form a growing primer strand via a polymerase reaction; (v) imaging the growing primer strand to identify the incorporated compound of step (iv) via its reporter; (vi) exposing the solid surface with the growing primer strand to a light source to remove a photocleavable terminating moiety of the formula: with the variables as defined by the claim referenced in step (iii); and (vii) repeating steps (iv) through (vi) one or more times to identify a plurality of bases in the target nucleic acid, where the extended primer of step (vi) of the previous cycle reacts in place of the hybridized primer/target nucleic acid complex in step (iv) of the subsequent cycle. |
