Composition for 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.
1 . A composition comprising a polymerizing agent including at least one molecular and/or atomic tag located at or near the polymerizing agent or associated with the polymerizing agent, a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form an extendable primer-template duplex, and monomers for the polymerizing agent, where a detectable property of the tag undergoes a change before, during and/or after one or a plurality of monomer incorporations.
2 . The composition of claim 1 , wherein the detectable property has a first value when the polymerizing agent is in a first state and a second value when the polymerase is in a second state, and where the polymerizing agent changes from the first state to the second state and back again during one monomer incorporation or a plurality of monomer incorporations.
3 . The composition of claim 2 , wherein the polymerizing agent is a polymerase or reverse transcriptase.
4 . The composition of claim 3 , 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.
5 . The composition of claim 3 , wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.
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7 . A composition comprising a polymerase or reverse transcriptase including at least one molecular and/or atomic tag located at or near the polymerizing agent or associated with the polymerase or reverse transcriptase, where a detectable property has a first value when the polymerase or reverse transcriptase is in a first state and a second value when the polymerase or reverse transcriptase is in a second state during monomer incorporation, and where the polymerase or reverse transcriptase changes from the first state to the second state and back again during each monomer incorporation.
8 . The composition of claim 7 , 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.
9 . The composition of claim 7 , wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.
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35 . The composition of claim 1 , wherein the monomers comprise nucleotides and the monomer tags are covalently bonded to a β, γ or terminal phosphate group of the tagged nucleotide types.
36 . The composition of claim 35 , wherein the detectable property is a fluorescent property comprising a duration, an intensity and/or frequency of emitted light.
37 . The composition of claim 36 , wherein each monomer type includes a fluorescent tag, and each fluorescent tag is the same or different.
38 . The composition of claim 35 , wherein at least two monomer types include a fluorescent tag, and each fluorescent tag is the same or different.
39 . The composition of claim 35 , wherein at least three monomer types include a fluorescent tag, and each fluorescent tag is the same or different.
40 . The composition of claim 1 , further comprising:
a collisional quencher adapted to quench fluorescence of the tagged monomers in the medium.
41 . A composition comprising:
a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent or associated with the polymerizing agent,
a template,
a primer, and
monomers for the polymerizing agent, where one monomer type includes a monomer fluorescent tag covalently bonded to a part of the monomer that is release during monomer incorporation,
where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged monomer incorporations due to an interaction between the polymerizing agent tag and the monomer tag.
42 . The composition of claim 41 , wherein the polymerizing agent is a polymerase or reverse transcriptase.
43 . The composition of claim 42 , 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 wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.
44 . The composition of claim 43 , wherein the polymerase comprises Taq DNA polymerase I having a tag attached to an amino acid at a specific amino acid position of the Taq DNA polymerase I, where the amino acid position is selected from the group consisting of 513-518, 643, 647, 649 and 653-661 of SEQ. ID No. 11.
45 . The composition of claim 41 , wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.
46 . The composition of claim 41 , wherein the monomer tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the monomer tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged monomer incorporation.
47 . The composition of claim 46 , wherein at least two monomer types include fluorescent tags, where the tags for each monomer type are the same or different and each monomer tag form a FRET pair with the polymerizing agent tag.
48 . The composition of claim 46 , wherein at least three monomer types include fluorescent tags, where the tags for each monomer type are the same or different and each monomer tag form a FRET pair with the polymerizing agent tag.
49 . The composition of claim 46 , wherein each monomer types include fluorescent tags, where the tags for each monomer type are the same or different and each monomer tag form a FRET pair with the polymerizing agent tag.
50 . The composition of claim 41 , further comprising:
a collisional quencher adapted to quench the fluorescent of the tagged monomers in the medium.
51 . A composition comprising:
a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent or associated with the polymerizing agent,
a template,
a primer, and
nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the β terminal phosphate group thereof,
where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.
52 . The composition of claim 51 , wherein the polymerizing agent is a polymerase or reverse transcriptase.
53 . The composition of claim 51 , wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.
54 . The composition of claim 51 , wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.
55 . The composition of claim 54 , wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
56 . The composition of claim 54 , wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
57 . The composition of claim 54 , wherein each dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
58 . The composition of claim 51 , further comprising:
a collisional quencher adapted to quench the fluorescent of the tagged dNPTs in the medium.
59 . A composition comprising:
a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the polymerizing agent,
a template,
a primer, and
nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the γ phosphate group thereof,
where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.
60 . The composition of claim 59 , wherein the polymerizing agent is a polymerase or reverse transcriptase.
61 . The composition of claim 59 , wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.
62 . The composition of claim 59 , wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.
63 . The composition of claim 62 , wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
64 . The composition of claim 62 , wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
65 . The composition of claim 62 , wherein each dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
66 . The composition of claim 59 , further comprising:
a collisional quencher adapted to quench the fluorescent of the tagged dNPTs in the medium.
67 . A composition comprising:
a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the polymerizing agent,
a template,
a primer, and
nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the terminal phosphate group thereof,
where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.
69 . The composition of claim 67 , wherein the polymerizing agent is a polymerase or reverse transcriptase.
70 . The composition of claim 67 , wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.
71 . The composition of claim 67 , wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.
72 . The composition of claim 71 , wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
73 . The composition of claim 71 , wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
74 . The composition of claim 71 , wherein each dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
75 . The composition of claim 67 , further comprising:
a collisional quencher adapted to quench the fluorescent of the tagged dNPTs in the medium.
76 . A composition comprising:
a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent or associated with the polymerizing agent,
a template,
a primer, and
nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a dNTP fluorescent tag covalently bonded to a β and/or γ phosphate thereof,
where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.
77 . The composition of claim 76 , wherein the polymerizing agent is a polymerase or reverse transcriptase.
78 . The composition of claim 77 , 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 wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.
79 . The composition of claim 78 , wherein the polymerase comprises Taq DNA polymerase I having a tag attached to an amino acid at a specific amino acid position of the Taq DNA polymerase I, where the amino acid position is selected from the group consisting of 513-518, 643, 647, 649 and 653-661 of SEQ. ID No. 11.
80 . The composition of claim 76 , wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.
81 . The composition of claim 76 , wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.
82 . The composition of claim 81 , wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
83 . The composition of claim 81 , wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
84 . The composition of claim 81 , wherein each dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.
85 . The composition of claim 76 , further comprising:
a collisional quencher adapted to quench the fluorescent of the tagged dNTPs in the medium.