| 主权项 |
1. A method of polynucleotide sequencing comprising the steps of:
forming a mixture of a first, a second, a third, and a fourth class of polynucleotides such that: each polynucleotide in the first class includes a 3′-terminal dideoxyadenosine labeled with a first dye; each polynucleotide in the second class includes a 3′-terminal dideoxycytidine and is labeled with a second dye; each polynucleotide in the third class includes a 3′-terminal dideoxyguanosine and is labeled with a third dye; and each polynucleotide in the fourth class includes a 3′-terminal dideoxythymidine and is labeled with a fourth dye; wherein one of the first, second, third or fourth dyes is an extended rhodamine dye having a structure of one of the following formulae: wherein R1, R2, R3, R4, R5, R6, R7, R9, R10, R11, and R13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z1, halogen, —OS(O)2OR, —S(O)2OR, —S(O)2R, —S(O)2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —C(O)OR, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, and —OR; or R1 taken together with R2, Y1, or Y2; or R4 taken together with R3, Y3, or Y4; or R5 taken together with R6, Y3, or Y4; or R6 taken together with R7, Y3, or Y4; or R10 taken together with R9 or R11; or R11 taken together with Y1, or Y2; or R13 taken together with Y3 or Y4 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1; R8is selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1; Y1, Y2, Y3, Y4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1; or Y1 taken together with R1, R11 or Y2; or Y2 taken together with R1, R11 or Y1; or Y3 taken together with R4, R5, R6, R13 or Y4; or Y4 taken together with R4, R5, R6, R13 or Y3 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1; Z1is selected from —R, halogen, —OS(O)2OR, —SO2OR, —SO2R, —SO2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —CO2R, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linkage to the polynucleotide; and R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and a linkage to the labeled polynucleotide wherein the linkage is attached to the extended rhodamine compound at R1, R4, R5, R6, R7, R8, R9, R10, Y1, Y2, Y3, or Y4; and wherein each of the other of the four dyes is spectrally resolvable from the extended rhodamine dye and from each other; electrophoretically separating the labeled polynucleotides thereby forming bands of similarly sized polynucleotides; illuminating the bands with an illumination beam capable of causing the dyes to fluoresce; and identifying the classes of the labeled polynucleotides in the bands by the fluorescence spectrum of the dyes. |
