Polymerase-template complexes

Abstract

The present disclosure provides methods and compositions for enhancing the processivity of a polymerase in catalyzing template-dependent DNA synthesis in high concentrations of salt. Also disclosed are methods and compositions for enhancing the assembly of polymerase-template complex compatible with active DNA synthesis in the presence of low levels of nucleotides and at a high temperature, such as temperatures at or near the melting temperature of the polymerase.

What is claimed is: 
     
         1 . A method for preparing a polymerase-template complex, comprising:
 (a) providing a polymerase; and   (b) contacting the polymerase with a polynucleotide template in a solution comprising a low concentration of nucleotides and a high temperature, thereby preparing the polymerase-template complex.   
     
     
         2 . The method of  claim 1 , further comprising saturating the solution with the polymerase of the polymerase-template complex. 
     
     
         3 . The method of  claim 1 , wherein the concentration of nucleotides is 0.8 μM to 2.2 μM. 
     
     
         4 . The method of  claim 1 , wherein the high temperature of the solution is a temperature above room temperature. 
     
     
         5 . The method of  claim 4 , wherein the temperature of the solution is 35° C. to 45° C. 
     
     
         6 . The method of  claim 1 , further comprising raising the concentration of nucleotides in the solution after preparing the polymerase-template complex. 
     
     
         7 . The method of  claim 1 , wherein the polymerase of the polymerase-template complex has at least 85%, 90%, 95%, 98% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         8 . A method for increasing processivity of a template-polymerase complex, the method comprising
 forming a polymerase-template complex in a solution comprising a low concentration of nucleotides and a high temperature, wherein the processivity of the polymerase-template complex formed in the high-temperature solution is greater than a processivity resulting from a control polymerase-template complex solution at room temperature.   
     
     
         9 . The method of  8 , wherein the concentration of nucleotides is about 1.2 μM. 
     
     
         10 . The method of  claim 8 , wherein the temperature of the solution is about 40° C. 
     
     
         11 . The method of  claim 8 , further comprising raising the concentration of nucleotides in the solution after forming the polymerase-template complex. 
     
     
         12 . The method of  claim 8 , wherein the polymerase of the polymerase-template complex has at least 85%, 90%, 95%, 98% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         13 . A method for nanopore-based sequencing of a polynucleotide template, the method comprising:
 forming a polymerase-template complex in a solution comprising a low concentration of nucleotides, the solution having a high temperature;   combining the formed polymerase-template complex with a nanopore to form a nanopore-sequencing complex;   providing tagged nucleotides to the nanopore sequencing complex to initiate template-dependent nanopore sequencing of the template at the high temperature;   detecting, with the aid of the nanopore, a tag associated with each of the tagged nucleotides during incorporation of each of the tagged nucleotides while each of the tagged nucleotides is associated with the polymerase, thereby determining the sequence of the polynucleotide template.   
     
     
         14 . The method of  claim 13 , wherein forming the polymerase-template complex comprises saturating the solution with the polymerase of the polymerase-template complex. 
     
     
         15 . The method of  claim 13 , wherein the concentration of nucleotides is 0.8 μM to 2.2 μM. 
     
     
         16 . The method of  15 , wherein the concentration of nucleotides is 1.2 μM. 
     
     
         17 . The method of  claim 13 , wherein the temperature of the solution is about 40° C. 
     
     
         18 . The method of  claim 13 , wherein the polymerase of the polymerase-template complex has at least 85%, 90%, 95%, 98% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         19 . The method of  claim 13 , wherein the nanopore is an oligomeric nanopore. 
     
     
         20 . The method of  claim 19 , wherein the nanopore is an alpha-hemolysin nanopore.