Polypeptides having nucleic acid binding activity and compositions and methods for nucleic acid amplification

• US 20060228726A1
• Filed: 01/06/2006
• Published: 10/12/2006
• Est. Priority Date: 01/06/2005
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Simpson, David A., Hardy, Christine D., Martin, Patrick K.
• Application Number:
  US11/327,845
• Publication Number:
  US 20060228726A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2563/119 the label being proteinic

Abstract

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.

Claims

1. A method of amplifying a nucleic acid sequence, wherein the method comprises subjecting a reaction mixture to at least one amplification cycle, wherein the reaction mixture comprises a double-stranded nucleic acid, at least two primers capable of annealing to complementary strands of the double-stranded nucleic acid, and a fusion protein comprising a thermostable DNA polymerase and a nucleic acid binding polypeptide, and wherein the at least one amplification cycle comprises:

denaturing the double-stranded nucleic acid;

annealing the at least two primers to complementary strands of the denatured double-stranded nucleic acid; and

extending the at least two primers; and

wherein the time to complete one amplification cycle is 20 seconds or less.

4. The method of claim 1, wherein the annealing occurs at an annealing temperature that is greater than the predicted Tm of at least one of the primers.

5. The method of claim 4, wherein the annealing temperature is at least about 5°

C. greater than the predicted Tm of at least one of the primers.

6. The method of claim 4, wherein the annealing temperature is at least about 10°

C. greater than the predicted Tm of at least one of the primers.

10. The method of claim 4, wherein the extending occurs at the annealing temperature.

11. The method of claim 10, wherein the reaction mixture is held at the annealing temperature for 1 second or less.

12. The method of claim 10, wherein the denaturing occurs at a denaturing temperature that is sufficient to denature the double-stranded nucleic acid.

13. The method of claim 12, wherein the denaturing temperature is from about 85°

C. to about 100°

C.

14. The method of claim 12, wherein the reaction mixture is held at the denaturing temperature for 1 second or less.

15. The method of claim 14, wherein the reaction mixture is held at the denaturing temperature for 1 second or less and the annealing temperature for 1 second or less.

16. The method of claim 15, wherein the denaturing comprises bringing the reaction mixture to the denaturing temperature without holding the reaction mixture at the denaturing temperature after the denaturing temperature is reached, and bringing the reaction mixture to the annealing temperature without holding the reaction mixture at the annealing temperature after the annealing temperature is reached.

17. The method of claim 1, wherein the nucleic acid binding polypeptide comprises an amino acid sequence of a nucleic acid binding polypeptide from a thermophilic microbe.

18. The method of claim 17, wherein the nucleic acid binding polypeptide comprises an amino acid sequence of a nucleic acid binding polypeptide from Sulfolobus.

19. The method of claim 17, wherein the nucleic acid binding polypeptide is a Crenarchaeal nucleic acid binding polypeptide.

20. The method of claim 1, wherein the nucleic acid binding polypeptide comprises a sequence selected from:

a) SEQ ID NO;

20;

b) a sequence having at least 80% identity to SEQ ID NO;

20;

c) SEQ ID NO;

6;

d) a sequence having at least 80% identity to SEQ ID NO;

6;

e) SEQ ID NO;

1; and

f) a sequence having at least 80% identity to SEQ ID NO;

1.

21. The method of claim 1, wherein the thermostable DNA polymerase comprises an archaeal family B polymerase or a fragment or variant of an archaeal family B polymerase having polymerase activity.

22. The method of claim 21, wherein the thermostable DNA polymerase comprises Pfu polymerase or a fragment or variant of Pfu polymerase having polymerase activity.

23. The method of claim 21, wherein the reaction mixture further comprises a polypeptide having 5′

to 3′

exonuclease activity.

24. The method of claim 1, wherein the thermostable DNA polymerase comprises a bacterial family A polymerase or a fragment or variant of a bacterial family A polymerase having polymerase activity.

25. The method of claim 24, wherein the thermostable DNA polymerase comprises Taq DNA polymerase or a fragment or variant of Taq DNA polymerase having polymerase activity.

28. The method of claim 25, wherein the thermostable DNA polymerase comprises a variant of Taq DNA polymerase having increased processivity relative to naturally occurring Taq DNA polymerase.

2. (canceled)

3. (canceled)

7-9. -9. (canceled)

26. (canceled)

27. (canceled)

29-41. -41. (canceled)

42. A method of stabilizing an DNA:

RNA duplex comprising combining the DNA;

RNA duplex with a polypeptide comprising an amino acid sequence of a nucleic acid binding polypeptide or a fragment thereof having nucleic acid binding activity.

43-90. -90. (canceled)