Claims
- 1. A method of sequencing a sense strand and an antisense strand of a double-stranded polynucleotide comprising:
i) denaturing the double-stranded polynucleotide to provide the sense strand and the antisense strand; ii) reacting the sense strand with a first set of four differentially labeled polynucleotides; iii) reacting the antisense strand with a second set of four differentially labeled polynucleotides; iv) identifying each of the eight polynucleotides by a fluorescence or an absorption spectrum of the fluorophore; and v) determining the sequence of the sense strand from the polynucleotides differentially labeled with the first set of fluorophores and the sequence of the antisense strand from the polynucleotides differentially labeled with the second set of fluorophores.
- 2. The method of claim 1, wherein the fluorophore is at least one BODIPY fluorophore that has been chemically modified.
- 3. The method of claim 1, wherein said first set and second set of fluorophores are BODIPY 542/563, BODIPY B410, BODIPY B411, BODIPY 503/512, BODIPY 523/547, BODIPY 581/591, BODIPY 630/650, or BODIPY 650/665.
- 4. The method of claim 1, wherein said first set and second set of fluorophores are selected from the group consisting of fluoresceins, rhodamines, cyanines, coumarins, sulfonated pyrenes, squaraines and alexas.
- 5. The method of claim 1, wherein each fluorophore in the first set and the second set exhibits an adsorption maxima that is spectrally resolved as compared to the other fluorophores employed, and each fluorophore has an adsorption maxima in the range of about 500 to about 700 nm.
- 6. A method of 8-color sequencing of a polynucleotide comprising the steps of:
i) forming eight classes of polynucleotides wherein each class of polynucleotides is labeled with a fluorophore and each fluorophore is different; ii) electrophoretically separating the classes of polynucleotides; iii) illuminating the separated polynucleotides with a wavelength capable of causing the fluorophores to fluoresce; and iv) identifying the classes of polynucleotides by the fluorescence or absorption spectrum of the fluorophores.
- 7. The method of claim 6, wherein the fluorophores comprise at least one BODIPY fluorophore selected from the group consisting of BODIPY 542/563, BODIPY B410, BODIPY B411, BODIPY 503/512, BODIPY 523/547, BODIPY 581/591, BODIPY 630/650 and BODIPY 650/665.
- 8. The method of claim 6, wherein the fluorophores are selected from the group consisting of fluoresceins, rhodamines, cyanines, coumarins, sulfonated pyrenes, squaraines and alexas.
- 9. The method of claim 6 wherein each of the fluorophores exhibits a characteristic adsorption maxima that is spectrally resolved as compared to the other fluorophores in the set, and the adsorption maxima of each of the fluorophores in the set is in the range of about 500 to about 700 nm.
- 10. The method of claim 6, wherein the electrophoretically separating the polynucleotides takes place in at least one lane of the gel.
- 11. The method of claim 6, wherein the eight fluorophores are linked to the 5′ ends of the polynucleotides.
- 12. The method of claim 6, wherein the eight fluorophores are linked to the 3′ ends of the polynucleotides.
- 13. A method of distinguishing polynucleotides having different 3′-terminal dideoxynucleotides in a chain termination method of DNA sequencing, the method comprising the steps of:
i) forming eight classes of polynucleotides by extending from primers a plurality of polynucleotides by means of a DNA polymerase or a reverse transcriptase in the presence of a dideoxyadenosine triphosphate, a dideoxycytosine triphosphate, a dideoxyguanosine triphosphate, and a dideoxythymidine triphosphate, and wherein the eight classes of polynucleotides are labeled at a 5′ position with a different fluorophore; ii) electrophoretically separating the classes of polynucleotides; iii) illuminating the separated polynucleotides with a wavelength capable of causing the fluorophores to fluoresce; and iv) identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.
- 14. The method of claim 13, wherein said fluorophores comprise at least one BODIPY fluorophore selected from the group consisting of BODIPY 542/563, BODIPY B410, BODIPY B411, BODIPY 503/512, BODIPY 523/547, BODIPY 581/591, BODIPY 630/650 and BODIPY 650/665.
- 15. The method of claim 13, wherein said fluorophores are selected from the group consisting of fluoresceins, rhodamines, cyanines, coumarins, sulfonated pyrenes, squaraines and alexas.
- 16. The method of claim 13, wherein each of the fluorophores exhibits a characteristic adsorption maxima that is spectrally resolved as compared to the other fluorophores in the set, and the adsorption maxima is in the range of about 500 to about 700 nm.
- 17. The method of claim 13, wherein the DNA polymerase is Thermosequenase, AmpliTaqFS, Klenow fragment, SEQUENASE® DNA polymerase, Bst DNA polymerase, AMPLITAQ® DNA polymerase, Pfu (exo-)DNA polymerase, rTth DNA polymerase or Vent(exo-) DNA polymerase.
- 18. The method of claim 13, wherein the reverse transcriptase is AMV-RT, M-MuLV-RT or SuperScript RT.
- 19. The method of claim 13, wherein each BODIPY fluorophore in the set is coupled to the primer suitable for sequencing by a linker.
- 20. The method of claim 13, wherein each BODIPY fluorophore in the set is attached at the 5′ end of the products of the sequencing reaction and an additional fluorophore is attached at a 3′ position of the product of the sequencing reaction or at one or more internal positions of the products of the sequencing reaction.
- 21. The method of claim 13, wherein the additional fluorophore has an adsorption maxima of about 500 to about 700 nm and an emission maxima of about 500 to about 700 nm.
- 22. The method of claim 13, wherein the chain termination method of sequencing is performed by an automated DNA sequencing instrument.
- 23. A method for distinguishing polynucleotides having different ribonucleotides in a method of labeling polynucleotides by enzymatic incorporation, said method comprising the steps of:
i) forming a mixture of four classes of polynucleotides, the four classes comprising polynucleotides having different terminal nucleotide triphosphates, wherein said triphosphates are linked to a BODIPY fluorophore that contains at least one reactive functional group; and wherein said BODIPY fluorophores comprise a first set and all are different; ii) forming a second mixture of four classes of polynucleotides, the four classes comprising polynucleotides having different terminal nucleotide triphosphates; wherein said triphosphates are linked to a BODIPY fluorophore that contains at least one reactive functional group; and wherein said BODIPY fluorophores comprise a second set and all are different, and are different than the first set; iii) electrophoretically separating the polynucleotides in the first mixture and the second mixture; iv) illuminating the first and second mixtures with a wavelength capable of causing the fluorophores to fluoresce; and v) identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.
- 24. The method of claim 23, wherein said terminal nucleotide triphosphates are adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine triphosphate.
- 25. The method of claim 23, wherein said terminal nucleotide triphosphates are deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate.
- 26. The method of claim 23, wherein said terminal nucleotide triphosphates are dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, dideoxycytidine triphosphate, and dideoxythymidine triphosphate.
- 27. The method of claim 23, wherein said first set comprises at least one fluorophore selected from the group consisting of BODIPY 542/563, BODIPY B410, BODIPY B411, BODIPY 503/512, BODIPY 523/547, BODIPY 581/591, BODIPY 630/650 and BODIPY 650/665.
- 28. A method of labeling a nucleic acid for 8-color sequencing comprising the steps of:
i) forming a plurality of oligonucleotides substituted with at least two fluorophores comprising a donor and an acceptor; wherein said oligonucleotides are separated into eight classes, wherein said eight donor fluorophores comprise a donor set and are the same or different; and wherein eight acceptor fluorophores comprise an acceptor set and are all different; ii) annealing said oligonucleotide classes to a strand of a polymerase chain reaction product to generate a substrate for a 5′ to 3′ exonuclease activity; iii) amplifying said oligonucleotide classes, wherein said exonuclease activity degrades said oligonucleotides, wherein said donor is released; and iv) detecting said oligonucleotide classes.
- 29. The method of claim 28, wherein the acceptor fluorophore is different in all eight classes of oligonucleotides and the donor fluorophore is different or the same.
- 30. The method of claim 28, wherein said acceptor set and donor set comprise at least one BODIPY fluorophore selected from the group consisting of BODIPY 542/563, BODIPY B410, BODIPY B411, BODIPY 503/512, BODIPY 523/547, BODIPY 581/591, BODIPY 630/650 and BODIPY 650/665.
- 31. As a composition of matter, a 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-styryloxyacetate.
- 32. As a composition of matter, a 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-styryloxyacetate.
- 33. As a composition of matter, a 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Ser. No. 60/355,456, filed Feb. 5, 2002, hereby incorporated by reference in its entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60355456 |
Feb 2002 |
US |