SINGLE MOLECULE SEQUENCING WITH TWO DISTINCT CHEMISTRY STEPS
First Claim
1. A method for sequencing a nucleic acid template comprising:
- providing a sequencing mixture comprising a polymerase enzyme, a template nucleic acid, a primer, a polymerase regent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, a plurality of types of nucleotide analogs, wherein each type of nucleotide analog has a labeled leaving group, each with a different label, that blocks further nucleotide incorporations at the 3′
hydroxyl position of the incorporated nucleotide analog; and
a selective cleaving activity which more effectively cleaves a leaving group from an incorporated nucleotide analog than from an unincorporated nucleotide analog;
carrying out nucleic acid synthesis such that a plurality of nucleotide analogs are added sequentially whereby;
i), a nucleotide analog is incorporated, then ii) the labeled leaving group on that nucleotide analog is cleaved by the selective cleaving activity, making the 3′
hydroxyl group available for a subsequent nucleotide analog addition; and
detecting light from the labels while the polymerase reaction is occurring to determine a sequence of the template nucleic acid.
1 Assignment
0 Petitions
Accused Products
Abstract
Methods, Compositions, and Systems are provided for nucleic acid sequencing where the sequential incorporation of nucleotides uses two distinct chemical steps. A plurality of nucleotide analogs, each having a labeled leaving group at its 3′ hydroxyl can be sequentially added to a growing strand in the presence of a selective cleaving activity that cleaves the 3′ hydroxyl leaving group preferentially after it has been incorporated. The selective cleaving agent can comprise an exonuclease activity, and the exonuclease activity can be a polymerase-associated exonuclease activity. Nucleotide analogs having labels on both a cleavable polyphosphate portion and on a 3′ hydroxyl leaving group can provide signals characteristic of nucleotide analog incorporation. Systems having illumination optics, collection optics, and substrates observe signals from the labels as they are being incorporated into a growing nucleic acid strand, allowing for the sequencing of template nucleic acids.
-
Citations
87 Claims
-
1. A method for sequencing a nucleic acid template comprising:
-
providing a sequencing mixture comprising a polymerase enzyme, a template nucleic acid, a primer, a polymerase regent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, a plurality of types of nucleotide analogs, wherein each type of nucleotide analog has a labeled leaving group, each with a different label, that blocks further nucleotide incorporations at the 3′
hydroxyl position of the incorporated nucleotide analog; and
a selective cleaving activity which more effectively cleaves a leaving group from an incorporated nucleotide analog than from an unincorporated nucleotide analog;carrying out nucleic acid synthesis such that a plurality of nucleotide analogs are added sequentially whereby;
i), a nucleotide analog is incorporated, then ii) the labeled leaving group on that nucleotide analog is cleaved by the selective cleaving activity, making the 3′
hydroxyl group available for a subsequent nucleotide analog addition; anddetecting light from the labels while the polymerase reaction is occurring to determine a sequence of the template nucleic acid. - View Dependent Claims (2, 3, 4, 8, 11, 15, 19, 20, 24)
-
-
5-7. -7. (canceled)
-
9-10. -10. (canceled)
-
13-14. -14. (canceled)
-
16-18. -18. (canceled)
-
21-23. -23. (canceled)
-
25. A method for sequencing a nucleic acid template comprising:
-
providing a sequencing mixture comprising a polymerase enzyme, a template nucleic acid, a primer, a polymerase regent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, a plurality of types of nucleotide analogs;
wherein each type of nucleotide analog has attached to its polyphosphate portion a first labeled leaving group comprising a first label, and attached to its 3′
hydroxyl a second labeled leaving group comprising a second label;
wherein each type of nucleotide analog has at least one different label from each other type of nucleotide analog; and
wherein the second labeled leaving group blocks further nucleotide analog additions, and a selective cleaving activity which selectively cleaves a leaving group from an incorporated nucleotide more effectively than cleaving an unincorporated nucleotide analog;carrying out nucleic acid synthesis such that a plurality of nucleotide analogs are added sequentially whereby;
i) a nucleotide analog is incorporated, resulting in release of the first labeled leaving group, then ii) the second labeled leaving group on that nucleotide analog is cleaved by the selective cleaving activity, making the 3′
hydroxyl group available for a subsequent nucleotide analog addition; anddetecting light from the labels while the polymerase reaction is occurring to determine a sequence of the template nucleic acid. - View Dependent Claims (26, 27, 42, 43, 44, 45, 48)
-
-
28-41. -41. (canceled)
-
46-47. -47. (canceled)
-
51. A method for sequencing a nucleic acid template comprising:
-
providing a sequencing mixture comprising a polymerase enzyme, a template nucleic acid, a primer, a polymerase regent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, a plurality of types of cyclic nucleotide analogs;
wherein each type of cyclic nucleotide analog comprises a ring that is formed by a labeled linker K1 and a cleavable group U, the linker attached to the 5′
polyphosphate portion of the nucleotide analog and to the cleavable group, and the cleavable group attached to the oxygen of the 3′
hydroxyl of the cyclic nucleotide analog, wherein each type of nucleotide analog has &
different label from each other type of cyclic nucleotide analog;carrying out nucleic acid synthesis such that a plurality of nucleotide analogs are added sequentially whereby;
i) acyclic nucleotide analog is incorporated into the growing strand, resulting in the opening of the ring between the alpha and beta phosphates of the 5′
polyphosphate portion such that the growing strand is attached to the alpha phosphate, and the beta phosphate comprises the end of a chain attached to the oxygen of the 3′
hydroxyl, then ii) the end of the chain comprising the beta phosphate reacts with the cleavable group U to cleave a labeled cyclic leaving group, and to release the 3′
hydroxyl, making the 3′
hydroxyl group available for a subsequent cyclic nucleotide analog addition; anddetecting light from the labels while the polymerase reaction is occurring to determine a sequence of the template nucleic acid. - View Dependent Claims (52)
-
-
53-60. -60. (canceled)
-
61. A system for sequencing a nucleic acid template comprising:
-
providing a sequencing mixture Comprising a polymerase enzyme, a template nucleic acid, a primer, a polymerase regent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, a plurality of types of nucleotide analogs;
wherein each type of nucleotide analog has a labeled leaving group, each with a different label, that blocks further nucleotide incorporations at the 3′
hydroxyl position of the incorporated nucleotide analog; and
a selective cleaving activity which more effectively cleaves a leaving group from an incorporated nucleotide analog than from an unincorporated nucleotide analog;a substrate onto which either the template nucleic acid or the polymerase enzyme is bound; an excitation light source directed to the portions on the substrate where the template nucleotide or polymerase enzyme is bound; a detector for detecting emitted light the labeled leaving groups while the polymerase reaction is occurring; and a computer to determine a sequence of the template nucleic acid using the detected light from the labeled leaving groups.
-
-
62-69. -69. (canceled)
-
70. A composition comprising a molecule of formula II:
-
71-77. -77. (canceled)
-
78. A composition comprising a compound Of the structure of Formula IV.
-
79-85. -85. (canceled)
-
86. A composition comprising a compound of Formula V.
-
87. (canceled)
Specification