Method for rapid base sequencing in DNA and RNA with two base labeling

Claims

What is claimed is:

1. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified by attachment of a first fluorescent dye which fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, while the remaining three bases found in DNA or RNA are each modified by attachment of a second fluorescent dye which fluoresces to fluoresce at a second wavelength of light characteristic of the remaining modified bases when excited using the chosen electromagnetic radiation;

(c) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream

(d) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(e) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(f) detecting the fluorescence at either the first wavelength of light or at the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

2. The method as described in claim 1, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

3. The method as described in claim 1, wherein said steps (a) through (f) are sequentially repeated two additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different first base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the remaining three bases when the modified bases are sequentially excited by the chosen electromagnetic radiation.

4. The method as described in claim 1, wherein the chosen first base and the remaining three bases are modified by attach fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

5. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified to have a first reactive center selected from the group consisting of amino and sulfhydro groups, while the remaining three bases found in DNA or RNA are each modified to have a second reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(d) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(e) attaching a first fluorescent dye to each first reactive center and a second fluorescent dye to each second reactive center, such that the first fluorescent dye fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, and the second fluorescent dye fluoresces at a second wavelength of light characteristic of the remaining three bases found in DNA or RNA when excited using the chosen electromagnetic radiation;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or at the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

6. The method as described in claim 5, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

7. The method as described in claim 5, wherein said steps (a) through (g) are sequentially repeated two additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different first base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the remaining three bases when the modified bases are sequentially excited by the chosen electromagnetic radiation.

8. The method as described in claim 5, wherein the fluorescent dyes are separated from each base by attachment of a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

9. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified by attachment of a first fluorescent dye which fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, and the remaining three bases found in DNA or RNA are each modified to have a reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(d) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(e) attaching a fluorescent dye to each reactive center such that the fluorescent dye fluoresces at a second wavelength of light characteristic of the three bases modified to have a reactive center when excited using the electromagnetic radiation;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

10. The method as described in claim 9, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

11. The method as described in claim 9, wherein said steps (a) through (g) are sequentially repeated two additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different first base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the remaining three bases when the modified bases are sequentially excited by the chosen electromagnetic radiation.

12. The method as described in claim 9, wherein the chosen first base and the remaining three types of bases are modified by attachment of fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

13. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified to have a reactive center selected from the group consisting of amino and sulfhydro groups, and the remaining three bases found in DNA or RNA are each modified by attachment of a first fluorescent dye which fluoresces at a first wavelength of light characteristic of the remaining modified bases when excited using a chosen spectrum of electromagnetic radiation;

(c) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(d) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(e) attaching a fluorescent dye to each reactive center such that the fluorescent dye fluoresces at a second wavelength of light characteristic of the chosen first base when excited using the electromagnetic radiation;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

14. The method as described in claim 13, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

15. The method as described in claim 13, wherein said steps (a) through (g) are sequentially repeated two additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different first base chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the remaining three bases when the modified bases are sequentially excited by the chosen electromagnetic radiation.

16. The method as described in claim 13, wherein the chosen first base and the remaining three bases are modified by attachment of fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

17. A method for DNA and RNA nucleotide base sequencing comprising the steps

(a) isolating a single fragment of DNA and RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base type selected from the group consisting of the four bases found in DNA or RNA is modified by attachment of a first fluorescent dye which fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, and a second chosen base type selected from the bases found in DNA or RNA is modified by attachment of a second fluorescent dye which fluoresces at a second wavelength of light characteristic of the second modified base when excited using the chosen electromagnetic radiation;

(c) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream

(d) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(e) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(f) detecting the fluorescence at either the first wavelength of light or at the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

18. The method as described in claim 17, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

19. The method as described in claim 17, wherein said steps (a) through (f) are sequentially repeated five additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different combination of first base and second base are chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the chosen second base when the modified bases are sequentially excited by the chosen electromagnetic radiation.

20. The method as described in claim 17, wherein the chosen first base and the remaining three bases are modified by attachment of fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

21. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified to have a first reactive center selected from the group consisting of amino and sulfhydro groups, and a chosen second base selected from the group of four bases found in DNA or RNA is modified to have a second reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(d) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(e) attaching a first fluorescent dye to each first reactive center and a second fluorescent dye to each second reactive center, such that the first fluorescent dye fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, and the second fluorescent dye fluoresces at a second wavelength of light characteristic of the second modified base when excited using the chosen electromagnetic radiation;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or at the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

22. The method as described in claim 21, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

23. The method as described in claim 21, wherein said steps (a) through (g) are sequentially repeated five additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different combination first base and second base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the second base when the modified bases are sequentially excited by the chosen electromagnetic radiation.

24. The method as described in claim 21, wherein the fluorescent dyes are separated from each base by attachment of a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

25. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified by attachment of a first fluorescent dye which fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, and a chosen second base is modified to have a reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(d) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(e) attaching a fluorescent dye to each reactive center such that the fluorescent dye fluoresces at a second wavelength of light characteristic of the chosen second base modified to have a reactive center when excited using the electromagnetic radiation;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

26. The method as described in claim 25, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

27. The method as described in claim 25, wherein said steps (a) through (g) are sequentially repeated five additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different combination of first base and second base is chosen for modification such that the chosen first base fluoresces at a different first wavelength or light than the second wavelength of fluorescence of the chosen second base when the modified bases are sequentially excited by the chosen electromagnetic radiation.

28. The method as described in claim 25, wherein the chosen first base and the chosen second base are modified by attachment of fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

29. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified to have a first reactive center selected from the group consisting of amino and sulfhydro groups, while the remaining three bases found in DNA or RNA are each modified to have a second reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching a first fluorescent dye to each first reactive center and a second fluorescent dye to each second reactive center, such that the first fluorescent dye fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, and the second fluorescent dye fluoresces at a second wavelength of light characteristic of the remaining three types of bases found in DNA or RNA when excited using the chosen electromagnetic radiation;

(d) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(e) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or at the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules.

30. The method as described in claim 29, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

31. The method as described in claim 29, wherein said steps (a) through (g) are repeated two additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different first base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the remaining three bases when the modified bases are sequentially excited by the chosen electromagnetic radiation.

32. The method as described in claim 29, wherein the fluorescent dyes are separated from each base by attachment of a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

33. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified by attachment of a first fluorescent dye which fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, and the remaining three bases found in DNA or RNA are each modified to have a reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching a fluorescent dye to each reactive center such that the fluorescent dye fluoresces at a second wavelength of light characteristic of the three bases modified to have a reactive center when excited using the electromagnetic radiation;

(d) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(e) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

34. The method as described in claim 33, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

35. The method as described in claim 33, wherein said steps (a) through (g) are sequentially repeated two additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different first base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the remaining three bases when the modified bases are sequentially excited by the chosen electromagnetic radiation,

36. The method as described in claim 33, wherein the chosen first base and the remaining three bases are modified by attachment of fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

37. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified to have a reactive center selected from the group consisting of amino and sulfhydro groups, and the remaining three bases found in DNA or RNA are each modified by attachment of a first fluorescent dye which fluoresces at a first wavelength of light characteristic of the remaining modified bases when excited using a chosen spectrum of electromagnetic radiation;

(c) attaching a fluorescent dye to each reactive center such that the fluorescent dye fluoresces at a second wavelength of light characteristic of the chosen first base when excited using the electromagnetic radiation;

(d) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(e) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

38. The method as described in claim 37, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

39. The method as described in claim 37, wherein said steps (a) through (g) are sequentially repeated two additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different first base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluoresce has of the remaining three bases when the modified bases are sequentially excited by the chosen electromagnetic radiation.

40. The method as described in claim 37, wherein the chosen first base and the remaining three bases are modified by attachment of fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

41. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified to have a first reactive center selected from the group consisting of amino and sulfhydro groups, and a chosen second base selected from the group of four bases found in DNA or RNA is modified to have a second reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching a first fluorescent dye to each first reactive center and a second fluorescent dye to each second reactive center, such that the first fluorescent dye fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, and the second fluorescent dye fluoresces at a second wavelength of light characteristic of the second modified base when excited using the chosen electromagnetic radiation;

(d) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(e) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or at the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

42. The method as described in claim 41, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

43. The method as described in claim 41, wherein said steps (a) through (g) are sequentially repeated five additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different combination first base and second base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the second base when the modified bases are sequentially excited by the chosen electromagnetic radiation.

44. The method as described in claim 41, wherein the fluorescent dyes are separated from each base by attachment of a linker am in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

45. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified by attachment of a first fluorescent dye which fluoresces at a first wavelength of light characteristic of the first modified base when excited using a chosen spectrum of electromagnetic radiation, and a chosen second base is modified to have a reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching a fluorescent dye to each reactive center such that the fluorescent dye fluoresces at a second wavelength of light characteristic of the chosen second base type modified to have a reactive center when excited using the electromagnetic radiation;

(d) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(e) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(f) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(g) detecting the fluorescence at either the first wavelength of light or the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

46. The method as described in claim 45, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

47. The method as described in claim 45, wherein said steps (a) through (g) are sequentially repeated five additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different combination of first base and second base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the chosen second base when the modified bases are sequentially excited by the chosen electromagnetic radiation.

48. The method as described in claim 45, wherein the chosen first base and the chosen second base are modified using fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

49. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified to have a first reactive center selected from the group consisting of amino and sulfhydro groups, and the remaining three bases found in DNA or RNA are each modified to have a second reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching a fluorescent dye to each first reactive center such that the fluorescent dye fluoresces at a first wavelength of light characteristic of the chosen first base modified to have a first reactive center when excited using a chosen spectrum of electromagnetic radiation;

(d) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(e) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(f) attaching a fluorescent dye to each second reactive center such that the fluorescent dye fluoresces at a second wavelength of light characteristic of the three bases modified to have a second reactive center when excited using the electromagnetic radiation;

(g) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(h) detecting the fluorescence at either the first wavelength of light or the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules.

50. The method as described in claim 49, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

51. The method as described in claim 49, wherein said steps (a) through (h) are sequentially repeated two additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different first base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the remaining three bases when the modified bases are sequentially excited by the chosen electromagnetic radiation.

52. The method as described in claim 49, wherein the chosen first base and the remaining three bases are modified by attachment of fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis add cleavage of the complementary strand of DNA or RNA.

53. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified to have a first reactive center selected from the group consisting of amino and sulfhydro groups, and the remaining three bases found in DNA or RNA are each modified to have a second reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching a fluorescent dye to each second reactive center such that the fluorescent dye fluoresces at a first wavelength of light characteristic of the three types of bases modified to have the second reactive center when excited using a chosen spectrum of electromagnetic radiation;

(d) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(e) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(f) attaching a fluorescent dye to each first reactive center such that the fluorescent dye fluoresces at a second wavelength of light characteristic of the chosen first base when excited using a chosen spectrum of electromagnetic radiation using the electromagnetic radiation;

(g) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(h) detecting the fluorescence at either the first wavelength of light or the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

54. The method as described in claim 53, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

55. The method as described in claim 53, wherein said steps (a) through (h) are sequentially repeated two additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different first base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the remaining three bases when the modified bases are sequentially excited by the chosen electromagnetic radiation.

56. The method as described in claim 53, wherein the chosen first base and the remaining three bases are modified by attachment of fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.

57. A method for DNA and RNA nucleotide base sequencing comprising the steps of:

(a) isolating a single fragment of DNA or RNA;

(b) enzymatically synthesizing a strand of DNA or RNA complementary to the single fragment of DNA or RNA to be sequenced using nucleotide bases modified such that a chosen first base selected from the group consisting of the four bases found in DNA or RNA is modified to have a first reactive center selected from the group consisting of amino and sulfhydro groups, and a chosen second base is modified to have a second reactive center selected from the group consisting of amino and sulfhydro groups;

(c) attaching a fluorescent 6lye to each first reactive center such that the fluorescent dye fluoresces at a first wavelength of light characteristic of the chosen first base modified to have a first reactive center when excited using a chosen spectrum of electromagnetic radiation

(d) attaching said synthesized complementary strand to a microsphere and suspending said microsphere in a laminar flow fluid stream;

(e) sequentially cleaving the end base from the DNA or RNA fragment to form a train of modified bases in said stream;

(f) attaching a fluorescent dye to each second reactive center such that the fluorescent dye fluoresces at a second wavelength of light characteristic of the chosen second base modified to have a second reactive center when excited using the electromagnetic radiation;

(g) exciting each modified base in the train of modified bases with the chosen electromagnetic radiation; and

(h) detecting the fluorescence at either the first wavelength of light or the second wavelength of light for each modified base in the train in sequential passage through a detector which detects the fluorescence from single molecules wherein said step of detecting provides information on the nucleotide sequence of said single fragment of DNA or RNA to be sequenced.

58. The method as described in claim 57, wherein said step of sequentially cleaving the end base from the DNA or RNA is achieved using exonuclease.

59. The method as described in claim 57, wherein said steps (a) through (h) are sequentially repeated five additional times using identical single fragments of DNA or RNA, whereby in each completed sequence of steps a different combination of first base and second base is chosen for modification such that the chosen first base fluoresces at a different first wavelength of light than the second wavelength of fluorescence of the chosen second base when the modified bases are sequentially excited by the chosen electromagnetic radiation.

60. The method as described in claim 57, wherein the chosen first base and the chosen second base are modified by attachment of fluorescent dyes which are separated from the base using a linker arm in order to assist in the synthesis and cleavage of the complementary strand of DNA or RNA.