Methods of using improved polymerases
AssigneeMJ Bioworks Incorporated
InventorsYan Wang, Peter Vander Horn, Lei Xi
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Abstract
This invention provides for methods of sequencing and performing polymerase reactions using an improved generation of nucleic acid polymerases. The improvement is the fusion of a sequence-non-specific nucleic-acid-binding domain to the enzyme in a manner that enhances the processivity of the polymerase.
Claims22
A method of increasing the yield from a polymerase reaction on a target nucleic acid present in a solution that comprises a polymerase inhibitor, the method comprising:
- (a) contacting the target nucleic acid with a polymerase, wherein the polymerase is joined to a sequence-non-specific nucleic-acid-binding domain that:
- (i) binds to double-stranded nucleic acid, and (ii) enhances the processivity of the polymerase compared to an identical polymerase not having the sequence non-specific nucleic-acid-binding domain fused to it, and
- wherein the solution is of a composition that permits the binding domain to bind to the target nucleic acid and the polymerase domain to extend a primer that is hybridized to the target nucleic acid sequence;
- (b) incubating the solution under conditions in which the primer is extended by the polymerase.
A method of sequencing a nucleic acid in an aqueous solution using an improved polymerase, the method comprising:
- (a) contacting the target nucleic acid with a polymerase, wherein the polymerase is joined to a sequence-non-specific nucleic-acid-binding domain that:
- (i) binds to double-stranded nucleic acid, and (ii) enhances the processivity of the polymerase compared to an identical polymerase not having the sequence non-specific nucleic-acid-binding domain fused to it, and
- wherein the solution is of a composition that permits the binding domain to bind to the target nucleic acid and the polymerase domain to extend a primer that is hybridized to the target nucleic acid sequence;
- (b) incubating the solution under conditions in which the primer is extended by the polymerase.
A method of claim 13, wherein the thermally stable polymerase domain is a ΔTaq polymerase domain.
A method of performing a quantitative polymerase reaction on a target nucleic acid present in a solution that comprises a DNA-binding fluorescent dye, the method comprising:
- (a) contacting the target nucleic acid with a polymerase, wherein the polymerase is joined to a sequence-non-specific nucleic-acid-binding domain that:
- (i) binds to double-stranded nucleic acid, and (ii) enhances the processivity of the polymerase compared to an identical polymerase not having the sequence non-specific nucleic-acid-binding domain fused to it, and
- wherein the solution is of a composition that permits the binding domain to bind to the target nucleic acid and the polymerase domain to extend a primer that is hybridized to the target nucleic acid sequence;
- (b) incubating the solution under conditions in which the primer is extended by the polymerase, and
- (c) exposing the solution to a suitable excitation light and measuring fluorescence emission from the fluorescent dye.
Dependencies are inferred from the claim text.