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US 9,309,561 B2Granted
Methods and kits using extended rhodamine dyes
AssigneeApplied Biosystems, LLC
InventorsJoe Y. L. Lam, Scott C. Benson, Steven M. Menchen
Life of this patent
Estimating the term…
Abstract
Extended rhodamine compounds exhibiting favorable fluorescence characteristics having the structure are disclosed. In addition, novel intermediates for synthesis of these dyes are disclosed, such intermediates having the structure In addition, methods of making and using the dyes as fluorescent labels are disclosed.
Claims12
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.
39 more elements
A method for sequencing a nucleic acid sequence comprising:
- forming a mixture of extended labeled primers by hybridizing a nucleic acid sequence with a fluorescently labeled oligonucleotide primer in the presence of deoxynucleotide triphosphates, at least one dideoxynucleotide triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleotide triphosphates until a dideoxynucleotide triphosphate is incorporated which terminates extension of the primer;
- separating the mixture of extended primers;
- and
- determining the sequence of the nucleic acid sequence by fluorescently measuring the mixture of extended primers formed;
- wherein the fluorescently labeled oligonucleotide primer comprises an extended rhodamine dye having a structure of the formula:
- 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;
- R8 is 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;
- Z1 is 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 fluorescently labeled primer;
- and
- wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and a linkage to the fluorescently labeled primer;
- 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 linkage is attached to the fluorescently labeled primer at a nucleobase of the primer:
- (a) when the nucleobase comprises a purine base, the linkage connecting the extended rhodamine dye to the nucleobase is attached to the 8-position of the purine base;
- (b) when the nucleobase comprises a 7-deazapurine base, the linkage connecting the extended rhodamine dye to the nucleobase is attached to the 7-position of the 7-deazapurine base;
- and
- (c) when the nucleobase comprises a pyrimidine base, the linkage connecting the extended rhodamine dye to the nucleobase is attached to the 5-position of the pyrimidine base.
37 more elements
Dependencies are inferred from the claim text.