Polymerase compositions and methods
Disclosed herein are modified polymerase compositions exhibiting altered polymerase activity, which can be useful in a variety of biological applications. Also disclosed herein are methods of making and using such compositions. In some embodiments, the compositions exhibit altered properties that can enhance their utility in a variety of biological applications. Such altered properties, can include, for example, altered nucleotide binding affinities, altered nucleotide incorporation kinetics, altered photostability and/or altered nanoparticle tolerance, as well as a range of other properties as disclosed herein.
1. A method for performing a primer extension reaction, comprising: contacting a modified polymerase comprising the amino acid sequence of SEQ ID NO: 7 with a nucleic acid molecule and a nucleotide under conditions where the nucleotide is incorporated into the nucleic acid molecule by the polymerase.
2. The method of claim 1 , wherein the nucleotide is a labeled nucleotide, and the label of the nucleotide emits a signal during incorporation of the nucleotide.
3. The method of claim 2 , further comprising detecting the signal emitted by the nucleotide label.
4. The method of claim 3 , further comprising analyzing the detected signal to determine the identity of the incorporated nucleotide.
5. The method of claim 1 , wherein the modified polymerase exhibits a ti value for a labeled nucleotide that is equal to or greater than the t −1 value of a reference Phi-29 polymerase comprising the amino acid sequence of SEQ ID NO: 1 for the same nucleotide.
6. The method of claim 1 , wherein the modified polymerase exhibits a t pol value for a labeled nucleotide that is equal to or greater than the t pol value of a reference Phi-29 polymerase comprising the amino acid sequence of SEQ ID NO: 1 for the same nucleotide.
7. The method of claim 1 , wherein the modified polymerase exhibits a residence time for a labeled nucleotide that is equal to or greater than the residence time of a reference Phi-29 polymerase comprising the amino acid sequence of SEQ ID NO: 1 for the same nucleotide.
8. The method of claim 1 , wherein the modified polymerase has a photostability of at least about 80% under standard photostability assay conditions.
9. The method of claim 1 , wherein the modified polymerase has a nanoparticle tolerance of at least about 80% under standard tolerance assay conditions.
10. The method of claim 2 , wherein the labeled nucleotide comprises a nucleotide label linked to the base, sugar or phosphate group of the nucleotide.
11. The method of claim 2 , wherein the labeled nucleotide is a reversible terminator.
12. The method of claim 2 , wherein the modified polymerase is linked to a label.
13. The method of claim 1 , wherein the modified polymerase further comprises a peptide tag which includes the amino acid sequence MNHLVHHHHHHIEGRHMELGTLEGS (SEQ ID NO:51), wherein the peptide tag is linked to the N-terminal or C-terminal end of the modified DNA polymerase.
14. The method of claim 1 , wherein the modified polymerase further comprises a peptide tag which includes the amino acid sequence MHHHHHHKH (SEQ ID NO:52), wherein the peptide tag is linked to the N-terminal or C-terminal end of the modified DNA polymerase.
15. The method of claim 1 , wherein the modified polymerase further comprises a peptide tag which includes the amino acid sequence LLGAAAKGAAAKGSAA (SEQ ID NO:16), wherein the peptide tag is linked to the N-terminal or C-terminal end of the modified DNA polymerase.
16. The method of claim 1 , wherein the modified polymerase further comprises a peptide tag which includes the amino acid sequence LLGGGGSGGGGSAAAGSAA (SEQ ID NO:17), wherein the peptide tag is linked to the N-terminal or C-terminal end of the modified DNA polymerase.
17. The method of claim 1 , wherein the modified polymerase further comprises a peptide tag which includes the amino acid sequence GLNDIFEAQKIEWHE (SEQ ID NO:53), wherein the peptide tag is linked to the N-terminal or C-terminal end of the modified DNA polymerase.