Polymerase-nucleic acid complex
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Publication Number
US-10000798-B2
Publication Date
2018-06-19
Expiration Date
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.
Core Innovation
The invention provides a polymerase-nucleic acid complex attached to a solid support, where a polymerase comprising a nucleic acid binding cleft is coupled to a target nucleic acid using a topological tether. The topological tether is attached to the polymerase through at least two anchors that straddle the nucleic acid binding cleft, thereby entraps the target nucleic acid in the nucleic acid binding cleft of the polymerase.
The complex further uses topological tether architecture that irreversibly associates the target nucleic acid with the nucleic acid binding cleft of the polymerase. In this arrangement, a circular target nucleic acid can be covalently attached to the polymerase through the topological tether via at least two anchors that straddle the nucleic acid binding cleft, with the tether irreversibly associating the circular target nucleic acid with the nucleic acid binding cleft.
The described framework includes polymerase-specific forms and additional components, including a DNA primer that complements a region of the target nucleic acid, as well as embodiments using phi29 polymerase and embodiments using a reverse transcriptase. The overall system is also described as a composition that includes detectably labeled nucleoside triphosphates with the polymerase-nucleic acid complex.
Claims Coverage
The independent claims cover polymerase-nucleic acid complexes and a related composition, centered on a topological tether coupled to a polymerase via at least two anchors that straddle the nucleic acid binding cleft. Across the set, the claims include seven inventive features.
Topological tether straddling the nucleic acid binding cleft
A 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.
Attachment to a solid support
The polymerase-nucleic acid complex is attached to a solid support.
Covalently attached topological tether for irreversible association
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.
Higher processivity index
The polymerase-nucleic acid complex shows a higher processivity index than a polymerase-nucleic acid complex without said topological tether.
DNA primer complements a region of the target nucleic acid
The polymerase-nucleic acid complex further comprises a DNA primer which complements a region of the target nucleic acid.
Phi29 polymerase with straddling anchors
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.
Reverse transcriptase with straddling anchors
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.
Composition with detectably labeled nucleoside triphosphates
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.
Collectively, the independent claims cover polymerase-nucleic acid complexes where a topological tether attached via at least two anchors straddles the nucleic acid binding cleft to entrap the target, including embodiments with an attached solid support, covalently attached tether for irreversible association and increased processivity index, circular DNA targets, a complementary DNA primer, and specific polymerase types (phi29 and reverse transcriptase). Claim clm-00026 additionally covers a composition that includes detectably labeled nucleoside triphosphates with such a polymerase-nucleic acid complex.
Stated Advantages
Shows a higher processivity index than a polymerase-nucleic acid complex without said topological tether.
Documented Applications
No documented applications found
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