Methods of using improved polymerases
First Claim
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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 thermally stable DNA polymerase, wherein the DNA polymerase is joined to a sequence non-specific double-stranded nucleic acid binding domain that specifically binds to polyclonal antibodies generated against either Sso7d (SEQ ID NO;
2) or Sac7d (amino acids 7-71 of SEQ ID NO;
10) 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).
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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.
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4 Claims
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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:
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(a) contacting the target nucleic acid with a thermally stable DNA polymerase, wherein the DNA polymerase is joined to a sequence non-specific double-stranded nucleic acid binding domain that specifically binds to polyclonal antibodies generated against either Sso7d (SEQ ID NO;
2) or Sac7d (amino acids 7-71 of SEQ ID NO;
10) 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)
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