Patent ID: 8198029

Claim:
A method of determining the sequence of a target nucleic acid comprising (i) adding a target nucleic acid to a Sanger or Sanger-type sequencing apparatus, (ii) adding one or more compounds of the formula: wherein R 1 =H, monophosphate, diphosphate or triphosphate, R 2 =H or OH, R 3 and R 4 are each independently selected from the group of H, a C 1 -C 12 straight chain or branched alkyl, a C 2 -C 12 straight chain or branched alkenyl or polyenyl, a C 2 -C 12 straight chain or branched alkynyl or polyalkynyl, and an aromatic group, R 5 , R 6 , and R 7 , are each independently selected from the group consisting of H, OCH 3 , NO 2 , CN, a halide, a C 1 -C 12 straight chain or branched alkyl, a C 2 -C 12 straight chain or branched alkenyl or polyenyl, a C 2 -C 12 straight chain or branched alkynyl or polyalkynyl, and an aromatic group, with the proviso that one of R 5 , R 6 , and R 7 is a group of the structure: wherein X═CH 2 , CH═CH, C≡C, O, S, or NH, Y═CH 2 , O, or NH, n=an integer from 0-12; m=an integer from 0-12, and Dye=a fluorophore, and R 8 and R 9 are as defined above for R 5 , R 6 , and R 7 , with the proviso that neither R 8 nor R 9 is NO 2 , to the sequencing apparatus, with the proviso that where more than one type of base is present, each base is attached to a different fluorophore; (iii) adding a complementary primer and a polymerase enzyme, (iv) performing a polymerase reaction to incorporate at least one of the compounds of step (ii) into a growing nucleic acid strand, and (v) analyzing the result of the Sanger sequencing reaction with fluorescence sequencing instrumentation or by pulsed multiline excitation fluorescence, wherein steps (i)-(iii) can be performed in any order.