Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores

Abstract

The invention relates to a new method of sequencing a double stranded target polynucleotide. The two strands of the double stranded target polynucleotide are linked by a bridging moiety. The two strands of the target polynucleotide are separated using a polynucleotide binding protein and the target polynucleotide is sequenced using a transmembrane pore.

1 .- 35 . (canceled) 
     
     
         36 . A method of single molecule sequencing and detecting a modified nucleotide base comprising:
 providing a membrane comprising a transmembrane pore inserted therein;   applying a voltage across the transmembrane pore;   controlling the movement of a single stranded polynucleotide through the transmembrane pore with a polynucleotide binding protein;   measuring the current passing through the transmembrane pore during each interaction with a nucleotide of the single stranded polynucleotide and obtaining information related to the rate the polynucleotide moves through the pore;   determining the sequence of the single-stranded polynucleotide as it moves through the pore based on changes in current flowing through the pore for a particular mean time period, and   determining the presence of the modified nucleotide base in the natural sequence based on a current signature produced in relation to movement of the polynucleotide through the pore at a rate controlled through contact of nucleotides of the polynucleotide with the polynucleotide binding protein.   
     
     
         37 . The method of  claim 36 , wherein the sequencing comprises reading the double stranded portion multiple times. 
     
     
         38 . The method of  claim 36 , wherein the polynucleotide binding protein comprises polymerase, exonuclease or helicase activity. 
     
     
         39 . The method of  claim 36 , wherein the polynucleotide binding protein is a DNA polymerase. 
     
     
         40 . The method of  claim 39 , wherein the DNA polymerase has 3′ to 5′ exonuclease activity. 
     
     
         41 . The method of  claim 36 , wherein the polynucleotide binding protein is a helicase. 
     
     
         42 . A method of single molecule sequencing and detecting a modified nucleotide base comprising:
 providing a membrane comprising a transmembrane pore inserted therein;   applying a voltage across the transmembrane pore;   controlling the movement of a single stranded polynucleotide through the transmembrane pore with a polynucleotide binding protein;   measuring the current passing through the transmembrane pore during each interaction with a nucleotide of the single stranded polynucleotide and obtaining information related to the rate the polynucleotide moves through the pore;   determining the sequence of the single-stranded polynucleotide as it moves through the pore based on changes in current flowing through the pore for a particular mean time period; and   determining the presence of the modified nucleotide base in the natural sequence based on a current signature produced in relation to movement of the polynucleotide through the pore at a rate controlled through contact of nucleotides of the polynucleotide with the polynucleotide binding protein,   wherein the single stranded polynucleotide comprises a natural DNA strand portion and a synthetic complement of the natural DNA strand portion.   
     
     
         43 . The method of  claim 42 , wherein the natural DNA strand portion is moved through the nanopore. 
     
     
         44 . The method of  claim 42 , wherein the synthetic complement of the natural DNA strand portion is moved through the nanopore. 
     
     
         45 . The method of  claim 42 , wherein the natural DNA strand portion and the synthetic complement of the natural DNA strand portion are attached through a hairpin loop and both portions are moved through the nanopore. 
     
     
         46 . The method of  claim 42 , wherein the polynucleotide binding protein comprise polymerase, exonuclease or helicase activity. 
     
     
         47 . The method of  claim 42 , wherein the polynucleotide binding protein is a DNA polymerase. 
     
     
         48 . The method of  claim 47 , wherein the DNA polymerase has 3′ to 5′ exonuclease activity. 
     
     
         49 . The method of  claim 42 , wherein the polynucleotide binding protein is a helicase. 
     
     
         50 . The method of  claim 42 , wherein the sequencing comprises reading the double stranded portion multiple times. 
     
     
         51 . The method of  claim 36 , wherein the membrane comprises a plurality of transmembrane pores 
     
     
         52 . The method of  claim 36 , wherein the single stranded polynucleotide comprises a natural DNA strand portion and a synthetic complement of the natural DNA strand portion. 
     
     
         53 . The method of  claim 52 , wherein the natural DNA strand portion is moved through the nanopore. 
     
     
         54 . The method of  claim 52 , wherein the synthetic complement of the natural DNA strand portion is moved through the nanopore. 
     
     
         55 . The method of  claim 52 , wherein the natural DNA strand portion and the synthetic complement of the natural DNA strand portion are attached through a hairpin loop and both portions are moved through the nanopore. 
     
     
         56 . A method for sequencing a nucleic acid template and identifying modified bases therein 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 processive enzyme associated with a template nucleic acid;   measuring the current through the nanopore over time as it is translated through the nanopore, wherein such measuring includes measuring the current and measuring the rate of translation;   determining the sequence of a portion of the template nucleic acid as it translates through the nanopore using the measured current over time; and   determining the presence of modified bases in the template nucleic acid by correlating changes in the rate of translation of the nucleic acid through the nanopore to changes in the kinetics of the processive enzyme from the interaction of the modified base with the processive enzyme.   
     
     
         57 . The method of  claim 56 , wherein the template nucleic acid is sequenced multiple times. 
     
     
         58 . The method of  claim 56 , wherein the processive enzyme comprises polymerase, exonuclease, or helicase activity. 
     
     
         59 . The method of  claim 56 , wherein the processive enzyme comprises a DNA polymerase. 
     
     
         60 . The method of  claim 59 , wherein the DNA polymerase has 3′ to 5′ exonuclease activity. 
     
     
         61 . The method  claim 56 , wherein the processive enzyme comprises a helicase. 
     
     
         62 . A method for sequencing a 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;   controlling the rate of translation of a single stranded portion of the template nucleic acid through the nanopore with a processive enzyme associated with a template nucleic acid;   measuring the current through the nanopore over time as it is translated through the nanopore, wherein such measuring includes measuring the current and measuring the rate of translation;   determining the sequence of a portion of the template nucleic acid as it translates through the nanopore using the measured current over time; and   determining the presence of modified bases in the template nucleic acid by correlating changes in the rate of translation of the nucleic acid through the nanopore to changes in the kinetics of the processive enzyme due to the interaction of the modified base with the processive enzyme,   wherein the template nucleic acid comprises hemi-genomic DNA comprising a genomic strand and a nascent strand.   
     
     
         63 . The method of  claim 62 , wherein the nascent strand is translated through the nanopore. 
     
     
         64 . The method of  claim 62 , wherein the genomic strand is translated through the nanopore. 
     
     
         65 . The method of  claim 62 , wherein the genomic strand and nascent strand are attached through a hairpin loop and both strands are translated through the nanopore. 
     
     
         66 . The method of  claim 62 , wherein the processive enzyme comprises polymerase, exonuclease, or helicase activity. 
     
     
         67 . The method of  claim 62 , wherein the processive enzyme comprises a DNA polymerase. 
     
     
         68 . The method of  claim 67 , wherein the DNA polymerase has 3′ to 5′ exonuclease activity. 
     
     
         69 . The method of  claim 62 , wherein the processive enzyme comprises a helicase. 
     
     
         70 . The method of  claim 62 , wherein the template nucleic acid is sequenced multiple times. 
     
     
         71 . The method of  claim 56 , wherein the substrate comprises from 1,000 to one million nanopores. 
     
     
         72 . The method of  claim 56 , wherein the template nucleic acid comprises hemi-genomic DNA comprising a genomic strand and a nascent strand. 
     
     
         73 . The method of  claim 72 , wherein the nascent strand is translated through the nanopore. 
     
     
         74 . The method of  claim 72 , wherein the genomic strand is translated through the nanopore. 
     
     
         75 . The method of  claim 72 , wherein the genomic strand and nascent strand are attached through a hairpin loop and both strands are translated through the nanopore. 
     
     
         76 . The method of  claim 36 , wherein the membrane is an amphiphilic layer, optionally a lipid bilayer. 
     
     
         77 . The method of  claim 42 , wherein the membrane is an amphiphilic layer, optionally a lipid bilayer. 
     
     
         78 . The method of  claim 36 , wherein the membrane separates a chamber into two sections, wherein a solution is present in each section. 
     
     
         79 . The method of  claim 42 , wherein the membrane separates a chamber into two sections, wherein a solution is present in each section.