Polymerase-nucleic acid complex
First Claim
1. A polymerase-nucleic acid complex attached to a solid support, the polymerase-nucleic acid complex comprising:
- a target nucleic acid, a polymerase comprising a nucleic acid binding cleft, and a topological tether, wherein the topological tether is attached to the polymerase through at least two anchors that straddle the nucleic acid binding cleft, whereby the topological tether entraps the target nucleic acid in the nucleic acid binding cleft of the polymerase, and wherein the polymerase-nucleic acid complex is attached to a solid support.
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Abstract
The present invention provides novel compositions, methods and apparatus for DNA sequencing that can be performed, e.g., in a two-electrode chamber. The present invention also provides a method for sequencing a nucleic acid comprising immobilizing a plurality of complexes comprising a target nucleic acid, a primer nucleic acid, and a polymerase onto a surface, contacting the surface with a plurality of charged particles comprising a nucleotide phosphate by applying an electric field, reversing the electric field to transport unbound charged particles away from the surface, and detecting the incorporation of a nucleotide phosphate into a single molecule of the primer nucleic acid.
28 Citations
26 Claims
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1. A polymerase-nucleic acid complex attached to a solid support, the polymerase-nucleic acid complex comprising:
a target nucleic acid, a polymerase comprising a nucleic acid binding cleft, and a topological tether, wherein the topological tether is attached to the polymerase through at least two anchors that straddle the nucleic acid binding cleft, whereby the topological tether entraps the target nucleic acid in the nucleic acid binding cleft of the polymerase, and wherein the polymerase-nucleic acid complex is attached to a solid support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A polymerase-nucleic acid complex, the polymerase-nucleic acid complex comprising:
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a circular target nucleic acid, a polymerase comprising a nucleic acid binding cleft, and a topological tether, wherein the topological tether is covalently attached to the polymerase through at least two anchors that straddle the nucleic acid binding cleft, thereby irreversibly associating the circular target nucleic acid with the nucleic acid binding cleft of the polymerase; and wherein said polymerase-nucleic acid complex shows a higher processivity index than a polymerase-nucleic acid complex without said topological tether.
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22. A polymerase-nucleic acid complex, the polymerase-nucleic acid complex comprising:
a target nucleic acid, a polymerase comprising a nucleic acid binding cleft, and a topological tether, wherein the topological tether is attached to the polymerase through at least two anchors that straddle the nucleic acid binding cleft, whereby the topological tether entraps the target nucleic acid in the nucleic acid binding cleft of the polymerase, and wherein the polymerase-nucleic acid complex further comprises a DNA primer which complements a region of the target nucleic acid.
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23. A polymerase-nucleic acid complex, the polymerase-nucleic acid complex comprising:
a target nucleic acid, a polymerase comprising a nucleic acid binding cleft, and a topological tether, wherein the topological tether is attached to the polymerase through at least two anchors that straddle the nucleic acid binding cleft, whereby the topological tether entraps the target nucleic acid in the nucleic acid binding cleft of the polymerase, and wherein the target nucleic acid is a circular DNA.
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24. A polymerase-nucleic acid complex, the polymerase-nucleic acid complex comprising:
a target nucleic acid, a phi29 polymerase comprising a nucleic acid binding cleft, and a topological tether, wherein the topological tether is attached to the phi29 polymerase through at least two anchors that straddle the nucleic acid binding cleft, whereby the topological tether entraps the target nucleic acid in the nucleic acid binding cleft of the phi29 polymerase.
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25. A polymerase-nucleic acid complex, the polymerase-nucleic acid complex comprising:
a target nucleic acid, a reverse transcriptase comprising a nucleic acid binding cleft, and a topological tether, wherein the topological tether is attached to the reverse transcriptase through at least two anchors that straddle the nucleic acid binding cleft, whereby the topological tether entraps the target nucleic acid in the nucleic acid binding cleft of the reverse transcriptase.
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26. A composition comprising a polymerase-nucleic acid complex and detectably labeled nucleoside triphosphates, the polymerase-nucleic acid complex comprising:
a target nucleic acid, a polymerase comprising a nucleic acid binding cleft, and a topological tether, wherein the topological tether is attached to the polymerase through at least two anchors that straddle the nucleic acid binding cleft, whereby the topological tether entraps the target nucleic acid in the nucleic acid binding cleft of the polymerase.
Specification