COMPOSITIONS, METHODS AND SYSTEMS FOR SINGLE MOLECULE SEQUENCING
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Abstract
Compositions, systems and methods of sequencing are disclosed, where the compositions and systems include polymerase enzymes that have been genetically modified to more efficiently incorporate nucleotides including labels having a detectable properties that are released during incorporation, to augment a rate of labeled nucleotide incorporation, to augment a rate of pyrophosphate release, or to augment two or more of these properties and rates. Also disclosed are terminally labeled and dual labeled nucleotides, and click-chemistry based methods of synthesizing the same.
136 Citations
67 Claims
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1-51. -51. (canceled)
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52. An isolated variant of Phi-29 polymerase comprising the amino acid sequence shown in SEQ ID NO:
- 3, wherein the variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;
3, and wherein the variant further comprises one or more mutations selected from the group consisting of;V250A/E375Y, V250A/E375A/Q380A, V250A/E375C, V250A/E375Y, V250I/E375A/Q380A, V250I/E375C, V250A, V250I, E375A, E375C, E375Y, E375A/Q380A, Q380A, D456N, D456E, D456S, D458N, V250A/E375A/Q380A/D456E, E375Y/V250L, E375Y/V250P, E375Y/V250Q, E375Y/V250R, E375Y/V250Y, E375Y/V250F, E375Y/V250S, E375Y/V250C, E375Y/V250T, E375Y/V250K, E375Y/V250H, E375Y/V250N, E375Y/V250D, E375Y/V250G, E375Y/V250W, E375Y/S388G, E375Y/K512A, E375Y/K525A, Y254V/E375Y, K132A, K383A, K383R, K383P, K371A, K371T, Y254F, Y254V, Y254S, Y254V, Y254S, K379A, K525A, K135A, P255S, S388G, K512A, L384R, E486A, E486D, K478A, E375W, N387A, N387Y, V250A/E375W, D456N/D458N/L351P, Y254V/A377E, D456N/D458N, D169A, D12A/D66A/D169A, T15I, N62D, C22S, C290S, C448S, C530S, C290S/C448S/C530S, C22S/C448S/C530S, C22S/C290S/C530S and C22S/C290S/C448S. - View Dependent Claims (65, 66, 67)
- 3, wherein the variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;
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53. An isolated variant of Phi-29 polymerase that comprises one or more mutations selected from the group consisting of:
- V250A/E375Y, V250A/E375A/Q380A, V250A/E375C, V250A/E375Y, V250I/E375A/Q380A, V250I/E375C, V250A, V250I, E375A, E375C, E375Y, E375A′
Q380A, Q380A, D456N, D456E, D456S, D458N, V250A/E375A/Q380A/D456E, E375Y/V250L, E375Y/V250P, E375Y/V250Q, E375Y/V250R, E375Y/V250Y, E375Y/V250F, E375Y/V250S, E375Y/V250C, E375Y/V250T, E375Y/V250K, E375Y/V250H, E375Y/V250N, E375Y/V250D, E375Y/V250G, E375Y/V250W, E375Y/S388G, E375Y/K512A, E375Y/K525A, Y254V/E375Y, K132A, K383A, K383R, K383P, K371A, K371T, Y254F, Y254V, Y254S, Y254V, Y254S, K379A, K525A, K135A, P255S, S388G, K512A, L384R, E486A, E486D, K478A, E375W, N387A, N387Y, V250A/E375W, D456N/D458N/L351P, Y254V/A377E, D456N/D458N, D169A, D12A/D66A/D169A, T15I, N62D, C22S, C290S, C448S, C530S, C290S/C448S/C530S, C22S/C448S/C530S, C22S/C290S/C530S and C22S/C290S/C448S. - View Dependent Claims (54, 55, 56, 57)
- V250A/E375Y, V250A/E375A/Q380A, V250A/E375C, V250A/E375Y, V250I/E375A/Q380A, V250I/E375C, V250A, V250I, E375A, E375C, E375Y, E375A′
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58-64. -64. (canceled)
Specification