The present disclosure relates to engineered DNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA polymerase polypeptides. The present disclosure also provides methods of using the engineered DNA polymerase polypeptides or compositions thereof for diagnostic and other purposes.

Provided herein include a method for modifying polymerase-nucleic acid complexes, including (a) providing a plurality of surface-immobilized polymerase-nucleic acid complexes in a vessel, wherein the nucleic acid includes a primed-template nucleic acid, wherein at least a subset of the surface-immobilized polymerase-nucleic acid complexes include ternary complexes further including nucleotides; and (b) washing the surface with an aqueous solution including a diol, sulfoxide or polyol, thereby removing the nucleotides from the vessel and retaining the surface-immobilized polymerase-nucleic acid complexes in the vessel.

Provided herein include a method for modifying polymerase-nucleic acid complexes, including (a) providing a plurality of surface-immobilized polymerase-nucleic acid complexes in a vessel, wherein the nucleic acid includes a primed-template nucleic acid, wherein at least a subset of the surface-immobilized polymerase-nucleic acid complexes include ternary complexes further including nucleotides; and (b) washing the surface with an aqueous solution including a diol, sulfoxide or polyol, thereby removing the nucleotides from the vessel and retaining the surface-immobilized polymerase-nucleic acid complexes in the vessel.

Disclosed are mutant DNA polymerases having increased 3′-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

Disclosed are mutant DNA polymerases having increased 3′-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

A method for identifying a nucleotide in a primed template nucleic acid, including the steps of (a) providing a vessel having a primed template nucleic acid, polymerase and a nucleotide cognate of a first base type; (b) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the first base type bound at a base position of the primed template nucleic acid; (c) delivering a nucleotide cognate of a second base type to the vessel, whereby the vessel retains the primed template nucleic acid and the polymerase from step (b); (d) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the second base type bound at the base position of the primed template nucleic acid; and (e) identifying the type of nucleotide at the base position of the primed template nucleic acid.

A method for identifying a nucleotide in a primed template nucleic acid, including the steps of (a) providing a vessel having a primed template nucleic acid, polymerase and a nucleotide cognate of a first base type; (b) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the first base type bound at a base position of the primed template nucleic acid; (c) delivering a nucleotide cognate of a second base type to the vessel, whereby the vessel retains the primed template nucleic acid and the polymerase from step (b); (d) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the second base type bound at the base position of the primed template nucleic acid; and (e) identifying the type of nucleotide at the base position of the primed template nucleic acid.

A method for amplifying a target nucleic acid including providing a system having a crRNA or a derivative thereof, and a Cas protein or a variant thereof. The crRNA or the derivative thereof contains a target-specific nucleotide region substantially complementary to a region of the target nucleic acid, and contacting the target nucleic acid with the system to form a complex.

A method for amplifying a target nucleic acid including providing a system having a crRNA or a derivative thereof, and a Cas protein or a variant thereof. The crRNA or the derivative thereof contains a target-specific nucleotide region substantially complementary to a region of the target nucleic acid, and contacting the target nucleic acid with the system to form a complex.

Embodiments may include a method of analyzing a nucleic acid molecule. The method may include attaching the nucleic acid molecule to a protein. The protein may be attached to a particle with a first diameter. The method may also include applying an electric field to move a first portion of the nucleic acid molecule into an aperture. The aperture may be defined by a first electrode, an insulator, and a second electrode. The aperture may have a second diameter less than the first diameter. The method may further include contacting the first portion of the nucleic acid molecule to both the first electrode and the second electrode. The method may include applying a voltage across the first electrode and the second electrode. The current through the electrodes and the portion of the nucleic acid molecule may be measured, and a nucleotide of the nucleic acid molecule may be identified.