Polypeptides having nucleic acid binding activity and compositions and methods for nucleic acid amplification
View Patent ↗Polypeptides having nucleic acid binding activity are provided. Methods of using polypeptides having nucleic acid binding activity are provided. Fusion proteins and methods of using fusion proteins are provided. Fusion proteins comprising a polymerase and a nucleic acid binding polypeptide are provided. Fusion proteins comprising a reverse transcriptase and a nucleic acid binding polypeptide are provided. Methods are provided for amplifying a nucleic acid sequence using a fusion protein comprising a nucleic acid binding polypeptide and a polymerase. Methods are provided for amplifying a nucleic acid sequence using a fusion protein comprising a nucleic acid binding polypeptide and a reverse transcriptase.
1. A recombinant fusion protein comprising:
(a) a first polypeptide having nucleic acid binding activity and comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 1, 4, or 6; and
(b) a second polypeptide having DNA polymerase activity and/or reverse transcriptase activity.
2. The fusion protein of claim 1 , wherein said second polypeptide further comprises 5′ to 3′ exonuclease activity.
3. The fusion protein of claim 1 , wherein said fusion protein exhibits an amplification efficiency that is from 0.8 to 1.0.
4. The fusion protein of claim 1 , wherein said first polypeptide and said second polypeptide are joined covalently.
5. The fusion protein of claim 1 , wherein said second polypeptide is thermostable.
6. The fusion protein of claim 1 , wherein said first polypeptide comprises the amino acid sequence set forth in SEQ ID NOs:1, 4, or 6.
7. The fusion protein of claim 1 , wherein said second polypeptide is an archaeal family B polymerase or a bacterial family A polymerase.
8. The fusion protein of claim 1 , wherein said second polypeptide is a polymerase selected from the group consisting of Taq polymerase, Pfu polymerase, Tgo polymerase, Vent polymerase, Deep Vent polymerase, KOD polymerase, and 9° Nm polymerase.
9. The fusion protein of claim 8 , wherein said polymerase is Pfu polymerase.
10. The fusion protein of claim 9 , wherein said Pfu polymerase is Pfu polymerase with a L→Y point mutation or a L→F point mutation at the position corresponding to the amino acid at position 409 of SEQ ID NO:30.
11. The fusion protein of claim 9 , wherein said Pfu polymerase is Pfu polymerase with a D→A point mutation at the position corresponding to the amino acid at position 141 of SEQ ID NO:30 and an E→A point mutation at the position corresponding to the amino acid at position 143 of SEQ ID NO:30.
12. The fusion protein of claim 9 , wherein said Pfu polymerase is Pfu polymerase with an E→D point mutation at the position corresponding to the amino acid at position 143 of SEQ ID NO:30.
13. The fusion protein of claim 9 , wherein said Pfu polymerase is Pfu polymerase with a Y→F point mutation at the position corresponding to the amino acid at position 385 of SEQ ID NO:30.
14. The fusion protein of claim 8 , wherein said polymerase is Taq polymerase.
15. A composition comprising:
(a) the recombinant fusion protein of claim 1 ; and
(b) a buffer.
16. The composition of claim 15 , further comprising an antibody that reversibly blocks said nucleic acid binding activity, DNA polymerase activity and/or reverse transcriptase activity at a first temperature.
17. The composition of claim 15 , further comprising an oligonucleotide that reversibly blocks said nucleic acid binding activity, DNA polymerase activity and/or reverse transcriptase activity at a first temperature.
18. The composition of claim 16 or 17 , wherein said antibody or said oligonucleotide specifically binds said first polypeptide and/or said second polypeptide.