Nanopore sequencing methods

Abstract

Methods are provided for sequencing of nucleic acid templates using nanopores. The rate of transport of the template nucleic acids through the nanopore is controlled using a polymerase enzyme having two slow kinetic steps. Methods are provided for sequencing hemi-natural nucleic acids such as hemi-genomic DNA, having two complementary strands, one a natural sequence and the other a synthetic sequence. The identification of modified bases can be enhanced by comparing the sequencing information from the natural sequence, which has, for example, natural base modifications, with the synthetic sequence, which typically has no base modifications. The presence and identity of a modified base can be determined by monitoring kinetics, for example the kinetics of polymer meditated nucleic acid synthesis.

What is claimed is: 
     
         1 . A method for sequencing a nucleic acid template comprising:
 providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a nanopore connecting the upper solution and lower solution, the nanopore sized to pass a single stranded nucleic acid;   providing a voltage across the nanopore to produce a measurable current flow through the nanopore;   controlling the rate of translation of a single stranded portion of the template nucleic acid through the nanopore with a polymerase enzyme that is associated with the template nucleic acid under conditions whereby the polymerase enzyme exhibits two kinetically observable steps;   measuring the current through the nanopore over time as the template nucleic acid is translated through the nanopore; and   determining the sequence of a portion of the template nucleic acid as it translates through the nanopore using the measured current over time.   
     
     
         2 . The method of  claim 1  wherein the two kinetically observable steps are selected from a group consisting of enzyme isomerization, nucleotide incorporation, and product release. 
     
     
         3 . The method of  claim 1  wherein the two kinetically observable steps are template translocation and nucleotide binding. 
     
     
         4 . The method of  claim 1  wherein the ratio of the rate constants of the kinetically observable steps is from 10:1 to 1:10. 
     
     
         5 . The method of  claim 1  wherein the rate constant for one of the kinetically observable steps is less than about 100 per second. 
     
     
         6 . The method of  claim 1  wherein the rate constant for one of the kinetically observable steps is between about 0.5 per second and about 60 per second. 
     
     
         7 . The method of  claim 1  wherein the polymerase enzyme comprises a modified recombinant Φ29-type polymerase. 
     
     
         8 . The method of  claim 7  wherein the polymerase enzyme comprises a modified recombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. 
     
     
         9 . The method of  claim 7  wherein the polymerase enzyme comprises a modified recombinant DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of E375Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L, K478Y, I370W, F198W, and L381A. 
     
     
         10 . The method of  claim 1  wherein the polymerase reaction conditions comprise one or more of metal cofactor concentration, pH, temperature, an enzyme activity modulator, D2O, an organic solvent, and buffer. 
     
     
         11 . The method of  claim 1  wherein the nucleic acid template comprises DNA. 
     
     
         12 . The method of  claim 1  wherein the template nucleic acid comprises a double stranded nucleic acid wherein the two strands are connected through a hairpin loop. 
     
     
         13 . The method of  claim 12  wherein one of the strands comprises a natural strand, and the other strand comprises a synthetic strand. 
     
     
         14 . The method of  claim 13  wherein the natural strand comprises genomic DNA. 
     
     
         15 . The method of  claim 13 , further comprising determining the presence of modified nucleic acids in the natural strand of the template nucleic acid by correlating changes in the rate of transport of the nucleic acid through the nanopore to the kinetics of the processive enzyme due to the interaction of the modified base with the polymerase enzyme. 
     
     
         16 . A method for sequencing a template nucleic acid comprising:
 providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a nanopore connecting the upper solution and lower solution, the nanopore sized to pass a single stranded nucleic acid;   providing a voltage across the nanopore to produce a measurable current flow through the nanopore;   providing a template nucleic acid comprising a double stranded nucleic acid having two strands are connected through a hairpin loop;   controlling the rate of translation of a single stranded portion of the template nucleic acid through the nanopore with a polymerase enzyme that is associated with the template nucleic acid under conditions whereby the polymerase enzyme exhibits two kinetically observable steps;   measuring the current through the nanopore over time as the template nucleic acid is translated through the nanopore; and   determining the sequence of a portion of the template nucleic acid as it translates through the nanopore using the measured current over time.   
     
     
         17 . The method of  claim 16  wherein the nucleic acid template comprises DNA. 
     
     
         18 . The method of  claim 16  wherein one of the strands comprises a natural strand, and the other strand comprises a synthetic strand. 
     
     
         19 . The method of  claim 18  wherein the natural strand comprises genomic DNA. 
     
     
         20 . The method of  claim 1 , further comprising determining the presence of modified nucleic acids in the natural strand of the template nucleic acid by correlating changes in the rate of transport of the nucleic acid through the nanopore to the kinetics of the processive enzyme due to the interaction of the modified base with the polymerase enzyme.