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

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.

1 - 90 . (canceled) 
     
     
         91 . A method of amplifying a nucleic acid sequence, wherein said method comprises subjecting a reaction mixture to at least one amplification cycle, wherein said reaction mixture comprises a double-stranded nucleic acid, at least two primers which anneal to complementary strands of said double-stranded nucleic acid, and a fusion protein comprising a thermostable DNA polymerase and a nucleic acid binding polypeptide, and wherein said at least one amplification cycle comprises:
 a) denaturing said double-stranded nucleic acid;   b) annealing said primers to complementary strands of said double-stranded nucleic acid; and   c) extending said primers for an amount of time between 2 to 10 seconds per thousand base pairs;   wherein said at least one amplification cycle is completed in 20 seconds or less and has an amplification efficiency of 0.8-1.0;   wherein said fusion protein produces an amount of amplification product that is greater than 10 fold higher than an amount of amplification product produced by said thermostable DNA polymerase alone, under the same conditions, and   wherein said fusion protein is reversibly blocked until a temperature is reached that reverses said blocking under amplification conditions.   
     
     
         92 . The method of  claim 91 , wherein said annealing occurs at a temperature greater than a predicted Tm of at least one of said primers. 
     
     
         93 . The method of  claim 91 , wherein said annealing and said extending occur at the same temperature. 
     
     
         94 . The method of  claim 91 , wherein said annealing and/or said denaturing is performed for one second or less. 
     
     
         95 . The method of  claim 91 , wherein said denaturing comprises incubation of said reaction mixture at a temperature from about 85° C. to about 100° C. 
     
     
         96 . The method of  claim 91 , wherein said denaturing comprises bringing said reaction mixture to a first temperature sufficient to denature said double-stranded nucleic acid without holding said reaction mixture at said first temperature for more than 1 second after said first temperature is reached, and said annealing comprises bringing said reaction mixture to a second temperature without holding said reaction mixture at said second temperature for more than 1 second after said second temperature is reached. 
     
     
         97 . The method of  claim 91 , wherein said nucleic acid binding polypeptide comprises an amino acid sequence of a nucleic acid binding polypeptide from a thermophilic microbe. 
     
     
         98 . The method of  claim 97 , wherein said thermophilic microbe is a  Sulfolobus  species. 
     
     
         99 . The method of  claim 91 , wherein said nucleic acid binding polypeptide is a Crenarchaeal nucleic acid binding polypeptide. 
     
     
         100 . The method of  claim 91 , wherein said nucleic acid binding polypeptide comprises an amino acid sequence selected from:
 a) SEQ ID NO:20;   b) an amino acid sequence having at least 80% identity to SEQ ID NO:20;   c) SEQ ID NO:6;   d) an amino acid sequence having at least 80% identity to SEQ ID NO:6;   e) SEQ ID NO:1; and   f) an amino acid sequence having at least 80% identity to SEQ ID NO:1.   
     
     
         101 . The method of  claim 91 , wherein said thermostable DNA polymerase comprises an archaeal family B polymerase or a fragment or variant of an archaeal family B polymerase having polymerase activity. 
     
     
         102 . The method of  claim 101 , wherein said thermostable DNA polymerase is derived from a Pfu polymerase. 
     
     
         103 . The method of  claim 91 , wherein said fusion protein further comprises a polypeptide having 5′ to 3′ exonuclease activity. 
     
     
         104 . The method of  claim 91 , wherein said thermostable DNA polymerase comprises a bacterial family A polymerase or a fragment or a variant of a bacterial family A polymerase having polymerase activity. 
     
     
         105 . The method of  claim 104 , wherein said thermostable DNA polymerase is derived from a Taq DNA polymerase. 
     
     
         106 . The method of  claim 105 , wherein said thermostable DNA polymerase is a variant of Taq DNA polymerase having increased processivity relative to a wild type Taq DNA polymerase. 
     
     
         107 . The method of  claim 91 , wherein said fusion protein is reversibly blocked by an antibody. 
     
     
         108 . The method of  claim 107 , wherein said antibody reversibly binds to said thermostable DNA polymerase portion of said fusion protein. 
     
     
         109 . The method of  claim 107 , wherein said antibody reversibly binds to said nucleic acid binding polypeptide portion of said fusion protein. 
     
     
         110 . The method of  claim 91 , wherein said fusion protein is reversibly blocked by a chemical modification. 
     
     
         111 . The method of  claim 91 , wherein said fusion protein is reversibly blocked by an inhibitory oligonucleotide. 
     
     
         112 . The method of  claim 91 , wherein said reaction mixture further comprises a reverse transcriptase (RT). 
     
     
         113 . The method of  claim 91 , wherein said reaction mixture further comprises Tween 20 and/or NP-40.