Methods of using improved polymerases
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.
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. The method of claim 1 , wherein the polymerase domain has thermally stable activity.
3. The method of claim 2 , wherein the polymerase domain comprises a ΔTaq polymerase domain.
4. The method of claim 2 , wherein the polymerase domain comprises a Pyrococcus polymerase domain.
5. The method of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2.
6. The method of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises the amino acid sequence set forth in SEQ ID NO:2, or Sac7d.
7. The method of claim 1 , wherein the dye is SYBR Green I.