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

Claim 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.

Claim 2

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.

Claim 6

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.

Claim 7

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.

Claim 11

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.

Claim 12

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.

Claim 16

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.

Claim 17

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.

Claim 21

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.

Claim 22

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.

Claim 26

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.

Claim 27

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.

Claim 31

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.

Claim 32

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.

Claim 36

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.

Claim 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 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.

Claim 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 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.

Claim 42

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.

Claim 46

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.

Claim 47

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.

Claim 51

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.

Claim 52

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.

Claim 56

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.

Claim 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 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.

Claim 61

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.

Claim 62

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.

Claim 66

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.

Claim 67

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.

Claim 71

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.

Claim 72

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.