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 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 which anneal 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:
 a) denaturing the double-stranded nucleic acid;   b) annealing the at least two primers to complementary strands of the denatured double-stranded nucleic acid; and   c) extending the at least two primers for an extension time to obtain primer extension products;   and wherein the time to complete one amplification cycle is 20 seconds or less and wherein the extension time is between 2 to 10 seconds per thousand base pairs of extension products.   
     
     
         92 . The method of  claim 91 , wherein the annealing occurs at an annealing temperature that is greater than the predicted Tm of at least one of the primers. 
     
     
         93 . The method of  claim 92 , wherein the annealing temperature is at least about 5° C. greater than the predicted Tm of at least one of the primers. 
     
     
         94 . The method of  claim 92 , wherein the annealing temperature is at least about 10° C. greater than the predicted Tm of at least one of the primers. 
     
     
         95 . The method of  claim 92 , wherein the extending occurs at the annealing temperature. 
     
     
         96 . The method of  claim 95 , wherein the reaction mixture is held at the annealing temperature for one second or less. 
     
     
         97 . The method of  claim 95 , wherein the denaturing occurs at a denaturing temperature that is sufficient to denature the double-stranded nucleic acid. 
     
     
         98 . The method of  claim 97 , wherein the denaturing temperature is from about 85° C. to about 100° C. 
     
     
         99 . The method of  claim 97 , wherein the reaction mixture is held at the denaturing temperature for 1 second or less. 
     
     
         100 . The method of  claim 99 , wherein the reaction mixture is held at the denaturing temperature for 1 second or less and the annealing temperature for 1 second or less. 
     
     
         101 . The method of  claim 100  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. 
     
     
         102 . The method of  claim 91 , wherein the nucleic acid binding polypeptide comprises an amino acid sequence of a nucleic acid binding polypeptide from a thermophilic microbe. 
     
     
         103 . The method of  claim 102 , wherein the nucleic acid binding polypeptide comprises an amino acid sequence of a nucleic acid binding polypeptide from Sulfolobus. 
     
     
         104 . The method of  claim 102 , wherein the nucleic acid binding polypeptide is a Crenarchaeal nucleic acid binding polypeptide. 
     
     
         105 . The method of  claim 91 , 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.   
     
     
         106 . The method of  claim 91 , 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. 
     
     
         107 . The method of  claim 106 , wherein the thermostable DNA polymerase comprises Pfu polymerase or a fragment or variant of Pfu polymerase having polymerase activity. 
     
     
         108 . The method of  claim 106 , wherein the reaction mixture further comprises a polypeptide having 5′ to 3′ exonuclease activity. 
     
     
         109 . The method of  claim 91 , 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. 
     
     
         110 . The method of  claim 109 , wherein the thermostable DNA polymerase comprises Taq DNA polymerase or a fragment or variant of Tag DNA polymerase having polymerase activity. 
     
     
         111 . The method of  claim 110 , wherein the thermostable DNA polymerase comprises a variant of Taq DNA polymerase having increased processivity relative to naturally occurring Taq DNA polymerase. 
     
     
         112 . The method of  claim 91 , wherein the extension time is 2 to 9 seconds per thousand base pairs of extension products. 
     
     
         113 . The method of  claim 91 , wherein the extension time is 2 to 8 seconds per thousand base pairs of extension products. 
     
     
         114 . The method of  claim 91 , wherein the extension time is 2 to 7 seconds per thousand base pairs of extension products. 
     
     
         115 . The method of  claim 91 , wherein the extension time is 2 to 6 seconds per thousand base pairs of extension products. 
     
     
         116 . The method of  claim 91 , wherein the extension time is 2 to 5 seconds per thousand base pairs of extension products. 
     
     
         117 . The method of  claim 91 , wherein the extension time is 2 to 4 seconds per thousand base pairs of extension products. 
     
     
         118 . The method of  claim 91 , wherein the extension time is 2 to 3 seconds per thousand base pairs of extension products. 
     
     
         119 . A method of generating DNA from RNA template comprising exposing the RNA template to at least one primer and a fusion protein comprising a nucleic acid binding polypeptide and a polymerase, wherein the polymerase is a family B polymerase, a fragment of a family B polymerase, or a polypeptide having at least 80% identity to a family of B polymerases, wherein the fusion protein has reverse transcriptase activity. 
     
     
         120 . A fusion protein comprising:
 a) a polypeptide comprising an amino acid sequence of a nucleic acid binding polypeptide or a fragment thereof having nucleic acid binding activity; and   b) a reverse transcriptase.