Method for increasing polymerase processivity

Abstract

The present invention provides methods for increasing polymerase processivity. In some aspects the invention includes providing a polymerase-nucleic acid complex having a nucleic acid comprising a template strand, and the template strand is entrapped in the polymerase through anchors that are covalently connected to each other across the nucleic acid binding cleft of the polymerase. In some cases the anchors comprise cysteines.

1 .- 20 . (canceled) 
     
     
         21 . A method of increasing polymerase processivity, the method comprising:
 providing a polymerase-nucleic acid complex, the polymerase-nucleic acid complex comprising:   a nucleic acid comprising a template strand, and   a polymerase comprising a nucleic acid binding cleft and at least two anchors that straddle the nucleic acid binding cleft; and   covalently connecting the at least two anchors to each other, thereby entrapping the template strand in the nucleic acid binding cleft of the polymerase.   
     
     
         22 . The method of  claim 21  wherein the at least two anchors comprise amino acids. 
     
     
         23 . The method of  claim 22  wherein at least one anchor comprises a cysteine 
     
     
         24 . The method of  claim 22  wherein the at least two anchors comprise a cysteine. 
     
     
         25 . The method of  claim 21  wherein the polymerase-nucleic acid complex comprises a primer. 
     
     
         26 . The method of  claim 21 , wherein the polymerase is selected from the group consisting of Klenow, Taq, phi29, RB-69, T4, and T7 polymerase. 
     
     
         27 . The method of  claim 21 , wherein the polymerase is phi29. 
     
     
         28 . The method of  claim 21 , wherein the polymerase is a DNA polymerase from  Thermus flavus, Pyrococcus furiosus, Thermotoga neapolitana, Thermococcus litoralis, Sulfolobus solfataricus, Thermatoga maritima , or  E. coli.    
     
     
         29 . The method of  claim 21 , wherein the polymerase is a reverse transcriptase. 
     
     
         30 . The method of  claim 21 , wherein the polymerase is an HIV reverse transcriptase. 
     
     
         31 . The method of  claim 21  further comprising carrying out nucleic acid synthesis using the polymerase-nucleic acid complex formed in  claim 21 . 
     
     
         32 . The method of  claim 31  wherein the polymerase-nucleic acid complex exhibits a processivity index of greater than 0.95 on a template 10,000 bases long or greater. 
     
     
         33 . The method of  claim 31 , wherein the nucleotides comprise detectably labeled nucleotides. 
     
     
         34 . The method of  claim 31 , wherein the nucleotides comprise detectably labeled nucleoside triphosphate. 
     
     
         35 . The method of  claim 21  wherein the template strand is circular DNA. 
     
     
         36 . The method of  claim 21  wherein the polymerase-nucleic acid complex is attached to a support.