Real-time sequence determination
A sequencing methodology is disclosed that allows a single DNA or RNA molecule or portion thereof to be sequenced directly and in substantially real time. The methodology involves engineering a polymerase and/or dNTPs with atomic and/or molecular tags that have a detectable property that is monitored by a detection system.
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25 . The apparatus of claims 24 , further comprising a plurality of monomer chambers, one for each tagged dNTP.
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34 . A single molecule sequencing apparatus comprising:
a reaction chamber including:
a polymerizing agent comprising a molecular fluorescent donor tag covalently bonded thereto and confined or immobilized within the reaction chamber, and a sequencing medium comprising an unknown nucleic acid template and a primer, where the primer is adapted to duplex to a portion of the template to form a primer-template complex and where the polymerizing agent and primer-template complex combine to form a sequencing complex,
a first deoxynucleotide triphosphate (dNTP) feed chamber including a first tagged dNTP having a first molecular fluorescent acceptor tag covalently bonded thereto,
a second deoxynucleotide triphosphate (dNTP) feed chamber including a second tagged dNTP having a second molecular fluorescent acceptor tag covalently bonded thereto,
a third deoxynucleotide triphosphate (dNTP) feed chamber including a third dNTP,
a fourth deoxynucleotide triphosphate (dNTP) feed chamber including a fourth dNTP,
channels connecting the dNTP feed chambers to the reaction chamber,
an excitation light source, and
a fluorescent detector/analyzer,
where the feed chambers are adapted to feed dNTPs to the reaction chamber, the light source is designed to irradiate the chamber with light of a wavelength sufficient to excite the donor tag on the polymerizing agent, and the detector/analyzer is designed to detect each fluorescent resonance energy transfer (FRET) event evidencing each incorporation of a tagged dNTP at the sequencing complex to produce a read out of each incorporation of a tagged dNTP.
35 . The apparatus of claim 34 , wherein the first acceptor tag and the second acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.
36 . The apparatus of claim 34 , wherein the third dNTP comprises a third tagged dNTP including a third molecular fluorescent acceptor tag covalently bonded thereto.
37 . The apparatus of claim 36 , wherein the first acceptor tag, the second acceptor tag and the third acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.
38 . The apparatus of claim 37 , wherein the fourth dNTP comprises a fourth tagged dNTP including a fourth molecular fluorescent acceptor tag covalently bonded thereto.
39 . The apparatus of claim 38 , wherein the first acceptor tag, the second acceptor tag, third acceptor tag and the fourth acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.
40 . The apparatus of claim 34 , wherein the third dNTP comprises a third tagged dNTP including a third molecular fluorescent acceptor tag covalently bonded thereto and the fourth dNTP comprises a fourth tagged dNTP including a fourth molecular fluorescent acceptor tag covalently bonded thereto.
41 . The apparatus of claim 40 , wherein the first acceptor tag, the second acceptor tag, third acceptor tag and the fourth acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.
42 . The apparatus of claim 34 , wherein the dNTPs are selected from the group consisting of dATP, dTTP, dCTP, and dGTP.
43 . The apparatus of claim 34 , wherein the dNTPs are selected from the group consisting of dATP, dTTP, dCTP, and dGTP and wherein each tag is covalently bonded to the γ-phosphate of its tagged dNTP.
44 . The apparatus of claim 34 , wherein the polymerizing agent is selected from the group consisting of a polymerase, a reverse transcriptase, and mixture or combinations thereof.
45 . The apparatus of claim 34 , wherein the polymerizing agent comprises a polymerase.
46 . The apparatus of claim 45 , wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I, and mixture or combinations thereof.
47 . The apparatus of claim 46 , wherein the polymerase has no 3′ to 5′ exonuclease activity.
48 . The apparatus of claim 34 , wherein the polymerizing agent comprises a reverse transcriptase.
49 . The apparatus of claim 48 , wherein the reverse transcriptase comprises human immunodeficiency virus reverse transcriptase (HIV-RT)
50 . The apparatus of claim 49 , wherein the reverse transcriptase has no 3′ to 5′ exonuclease activity.
51 . The apparatus of claim 34 , wherein the polymerizing agent comprising a naturally occurring or synthetic polypeptide and the polymerizing agent tag is covalently bonded to an SH group of a cysteine residue of the polypeptide, where the cysteine residue is located in a position on the polymerizing agent capable of supporting fluorescent resonance energy transfer (FRET) between the donor tag of the polymerizing agent and the acceptor tag on an incorporating tagged dNTP.
52 . The apparatus of claim 34 , wherein the template comprises a nucleic acid sequence from an organism including a human.
53 . The apparatus of claim 34 , wherein the template comprises a data stream.
54 . The apparatus of claim 34 , further comprising a plurality of polymerizing agents, a plurality of unknown templates, and a plurality of primers, where the unknown templates and the primers combine to form a plurality of primer-template complexes and where the polymerizing agents and primer-template complexes combine to form a sequencing complexes.
55 . The apparatus of claim 54 , wherein the third dNTP comprises a third tagged dNTP including a third molecular fluorescent acceptor tag covalently bonded thereto and the fourth dNTP comprises a fourth tagged dNTP including a fourth molecular fluorescent acceptor tag covalently bonded thereto.
56 . The apparatus of claim 55 , wherein the first acceptor tag, the second acceptor tag, third acceptor tag and the fourth acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.
57 . A single molecule sequencing apparatus comprising:
a reaction chamber including:
a substrate comprising a polymerizing agent comprising a molecular fluorescent donor tag covalently bonded thereto and immobilized on or to the substrate and,
a sequencing medium comprising an unknown nucleic acid template and a primer,
where the primer is adapted to duplex to a portion of the template to form a primer-template complex and where the polymerizing agent and primer-template complex combine to form a pre-sequencing complex,
a first deoxynucleotide triphosphate (dNTP) feed chamber including a first tagged dNTP having a first molecular fluorescent acceptor tag covalently bonded thereto,
a second deoxynucleotide triphosphate (dNTP) feed chamber including a second tagged dNTP having a second molecular fluorescent acceptor tag covalently bonded thereto,
a third deoxynucleotide triphosphate (dNTP) feed chamber including a third dNTP, a fourth deoxynucleotide triphosphate (dNTP) feed chamber including a fourth dNTP,
channels interconnecting the dNTP feed chambers with the reaction chamber,
an excitation light source, and
a fluorescent detector/analyzer,
where the feed chambers are adapted to feed dNTPs to the reaction chamber, the light source is designed to irradiate the chamber with light of a wavelength sufficient to excite the donor tag on the polymerizing agent, and the detector/analyzer is designed to detect each fluorescent resonance energy transfer (FRET) event evidencing each incorporation of a tagged dNTP at the sequencing complex to produce a read out of each incorporation of a tagged dNTP.
58 . The apparatus of claim 57 , wherein the first acceptor tag and the second acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.
59 . The apparatus of claim 57 , wherein the third dNTP comprises a third tagged dNTP including a third molecular and/or atomic fluorescent acceptor tag covalently bonded thereto.
60 . The apparatus of claim 59 , wherein the first acceptor tag, the second acceptor tag and the third acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.
61 . The apparatus of claim 60 , wherein the fourth dNTP comprises a fourth tagged dNTP including a fourth molecular and/or atomic fluorescent acceptor tag covalently bonded thereto.
62 . The apparatus of claim 61 , wherein the first acceptor tag, the second acceptor tag, third acceptor tag and the fourth acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.
63 . The apparatus of claim 57 , wherein the third dNTP comprises a third tagged dNTP including a third molecular and/or atomic fluorescent acceptor tag covalently bonded thereto and the fourth dNTP comprises a fourth tagged dNTP including a fourth molecular and/or atomic fluorescent acceptor tag covalently bonded thereto.
64 . The apparatus of claim 63 , wherein the first acceptor tag, the second acceptor tag, third acceptor tag and the fourth acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.
65 . The apparatus of claim 57 , wherein the dNTPs are selected from the group consisting of dATP, dTTP, dCTP, and dGTP.
66 . The apparatus of claim 57 , wherein the dNTPs are selected from the group consisting of dATP, dTTP, dCTP, and dGTP and wherein each tag is covalently bonded to the γ-phosphate of its tagged dNTP.
67 . The apparatus of claim 57 , wherein the polymerizing agent is selected from the group consisting of a polymerase, a reverse transcriptase, and mixture or combinations thereof.
68 . The apparatus of claim 67 , wherein the polymerizing agent comprises a polymerase.
69 . The apparatus of claim 68 , wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I, and mixture or combinations thereof.
70 . The apparatus of claim 69 , wherein the polymerase has no 3′ to 5′ exonuclease activity.
71 . The apparatus of claim 57 , wherein the polymerizing agent comprises a reverse transcriptase.
72 . The apparatus of claim 71 , wherein the reverse transcriptase comprises human immunodeficiency virus reverse transcriptase (HIV-RT)
73 . The apparatus of claim 72 , wherein the reverse transcriptase has no 3′ to 5′ exonuclease activity.
74 . The apparatus of claim 57 , wherein polymerizing agent comprising a naturally occurring or synthetic polypeptide and the polymerizing agent tag is covalently bonded to a cysteine residue of the polypeptide through a SH group of the cysteine residue, where the cysteine residue is located in a position on the polymerizing agent capable of supporting fluorescent resonance energy transfer from the donor tag of the polymerizing agent to the acceptor tags on incorporating tagged dNTPs.
75 . The apparatus of claim 57 , wherein the template comprises a nucleic acid sequence from an organism including a human.
76 . The apparatus of claim 57 , wherein the template comprises a data stream.
77 . The apparatus of claim 57 , further comprising a plurality of polymerizing agents, a plurality of unknown templates, and a plurality of primers, where the unknown templates and the primers combine to form a plurality of primer-template complexes and where the polymerizing agents and primer-template complexes combine to form a sequencing complexes.
78 . The apparatus of claim 77 , wherein the third dNTP comprises a third tagged dNTP including a third molecular fluorescent acceptor tag covalently bonded thereto and the fourth dNTP comprises a fourth tagged dNTP including a fourth molecular fluorescent acceptor tag covalently bonded thereto.
79 . The apparatus of claim 78 , wherein the first acceptor tag, the second acceptor tag, third acceptor tag and the fourth acceptor tag are different and their fluorescent emission spectra are detectably discernible one from the other.