Methods of using improved polymerases
First Claim
Patent Images
1. A method of performing a quantitative real-time polymerase chain 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 DNA polymerase, wherein the DNA polymerase is joined to a sequence non-specific double-stranded nucleic acid binding domain that comprises at least 75% amino acid sequence identity to the Sso7d amino acid sequence set forth in SEQ ID NO;
2 and enhances the processivity of the DNA polymerase compared to an identical DNA polymerase not having the sequence non-specific double-stranded nucleic-acid binding domain fused to it,wherein the solution comprises the DNA-binding fluorescent dye, which exhibits altered fluorescence emissions when the dye is bound to double-stranded DNA, and is of a composition that permits the sequence non-specific double-stranded nucleic acid 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 DNA polymerase;
(c) exposing the solution to a suitable excitation light and measuring fluorescence emission from the DNA-binding fluorescent dye; and
(d) performing at least one additional cycle of amplification comprising steps (a) to (c).
2 Assignments
0 Petitions
Accused Products
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.
9 Citations
7 Claims
-
1. A method of performing a quantitative real-time polymerase chain 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 DNA polymerase, wherein the DNA polymerase is joined to a sequence non-specific double-stranded nucleic acid binding domain that comprises at least 75% amino acid sequence identity to the Sso7d amino acid sequence set forth in SEQ ID NO;
2 and enhances the processivity of the DNA polymerase compared to an identical DNA polymerase not having the sequence non-specific double-stranded nucleic-acid binding domain fused to it,wherein the solution comprises the DNA-binding fluorescent dye, which exhibits altered fluorescence emissions when the dye is bound to double-stranded DNA, and is of a composition that permits the sequence non-specific double-stranded nucleic acid 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 DNA polymerase; (c) exposing the solution to a suitable excitation light and measuring fluorescence emission from the DNA-binding fluorescent dye; and (d) performing at least one additional cycle of amplification comprising steps (a) to (c). - View Dependent Claims (2, 3, 4, 5, 6, 7)
-
Specification