Methods and compositions for nanostructure-based nucleic acid sequencing

Abstract

Provided herein are nanostructure-based sequencing methods and systems.

1 . (canceled) 
     
     
         2 . A method of determining the sequence of a target nucleic acid molecule, comprising:
 providing a nanopore having a polymerase immobilized on or in the vicinity of said nanopore;   contacting a polymerase with a double-stranded naturally occurring target nucleic acid molecule under first sequencing conditions, wherein the first sequencing conditions comprise the presence of nucleoside triphosphates consisting of four nucleoside triphosphates that each lack a detectable label, wherein a first nucleoside triphosphate of the four nucleoside triphosphates is present in a rate-limiting amount;   detecting a pause in the movement of the target nucleic acid molecule and/or one or more nascent strand(s) through, on, or over the nanopore due to a pause in translocation of the target nucleic acid molecule and/or one or more nascent strand(s) by the polymerase;   repeating the contacting and detecting steps under second sequencing conditions or third sequencing conditions, both comprising the presence of nucleoside triphosphates consisting of four nucleoside triphosphates that each lack a detectable label, wherein a second nucleoside triphosphate of the four nucleoside triphosphates is present in a rate-limiting amount in the second sequencing conditions and wherein a third nucleoside triphosphate of the four nucleoside triphosphates is present in a rate-limiting amount in the third sequencing conditions; and   determining the sequence of the target nucleic acid molecule based on the pause(s) in the movement of the target nucleic acid molecule and/or one or more nascent strand(s) through, on, or over the nanopore detected under the first, second, and third sequencing conditions, wherein the pause(s) in movement indicate the presence of the rate-limiting nucleotide at that position.   
     
     
         3 . The method of  claim 2 , wherein the solid substrate is glass. 
     
     
         4 . The method of  claim 2 , wherein the polymerase is a RNA polymerase. 
     
     
         5 . The method of  claim 4 , wherein the RNA polymerase is selected from the group consisting of a bacteriophage RNA polymerase and a bacterial RNA polymerase. 
     
     
         6 . The method of  claim 5 , wherein the bacteriophage RNA polymerase is selected from the group consisting of a T7 RNA polymerase and a T3 RNA polymerase. 
     
     
         7 . The method of  claim 5 , wherein the bacterial RNA polymerase is an  E. coli  RNA polymerase. 
     
     
         8 . The method of  claim 2 , wherein the polymerase is a DNA polymerase. 
     
     
         9 . The method of  claim 8 , wherein the DNA polymerase is selected from the group consisting of phi29, T7 DNA polymerase,  Bacillus subtilis  DNA polymerase, and Taq DNA polymerase. 
     
     
         10 . The method of  claim 2 , wherein the target nucleic acid molecule further comprises a magnetic tag. 
     
     
         11 . The method of  claim 2 , wherein the detecting step comprises measuring a change in electric current of the nanopore. 
     
     
         12 . The method of  claim 2 , wherein the detecting step comprises measuring a change in ionic conduction of the nanopore. 
     
     
         13 . The method of  claim 2 , wherein the detecting step further comprises capturing movement on a CMOS based manufactured nanopore and electronics. 
     
     
         14 . The method of  claim 2 , further comprising:
 repeating the contacting and detecting steps under fourth sequencing conditions comprising the presence of nucleoside triphosphates consisting of four nucleoside triphosphates that each lack a detectable label, wherein a fourth nucleoside triphosphate of the four nucleoside triphosphates is present in a rate-limiting amount a plurality of times; and   determining the sequence of the target nucleic acid molecule based on the pause(s) in the movement of the target nucleic acid molecule and/or one or more nascent strand(s) through, on, or over the nanopore detected in the first, second, third, and fourth sequencing conditions.   
     
     
         15 . The method of  claim 2 , wherein the position in the target nucleic acid molecule is determined by detecting the cumulative amount of movement. 
     
     
         16 . A method of determining the sequence of a target nucleic acid molecule, comprising:
 providing a nanopore, wherein a polymerase is immobilized on or near the nanopore;   contacting the polymerase with the target nucleic acid molecule under first sequencing conditions comprising the presence of nucleoside triphosphates consisting of four nucleoside triphosphates, where a first nucleoside triphosphate of the four nucleoside triphosphates is present in a rate-limiting amount;   detecting a pause in the movement of the target nucleic acid molecule and/or one or more nascent strand(s) through, on, or over the nanopore due to a pause in translocation of the target nucleic acid molecule and/or one or more nascent strand(s) by the polymerase;   contacting the polymerase with the target nucleic acid molecule under second sequencing conditions comprising the presence of nucleoside triphosphates consisting of four nucleoside triphosphates, where a second nucleoside triphosphate of the four nucleoside triphosphates is present in a rate-limiting amount;   detecting a pause in the movement of the target nucleic acid molecule and/or one or more nascent strand(s) through, on, or over the nanopore due to a pause in translocation of the target nucleic acid molecule and/or one or more nascent strand(s) by the polymerase;   contacting the polymerase with the target nucleic acid molecule under third sequencing conditions comprising the presence of nucleoside triphosphates consisting of four nucleoside triphosphates, where a third nucleoside triphosphate of the four nucleoside triphosphates is present in a rate-limiting amount;   detecting a pause in the movement of the target nucleic acid molecule and/or one or more nascent strand(s) through, on, or over the nanopore due to a pause in translocation of the target nucleic acid molecule and/or one or more nascent strand(s) by the polymerase;   determining positional information of the first, second, and third nucleoside triphosphates along the target nucleic acid molecule based on the pause(s) in the movement of the target nucleic acid molecule and/or one or more nascent strand(s) through, on, or over the nanopore, wherein the pause(s) in movement indicate the presence of the rate-limiting nucleotide at that position.   
     
     
         17 . The method of  claim 16 , further comprising:
 contacting the polymerase with the target nucleic acid molecule under fourth sequencing conditions comprising the presence of nucleoside triphosphates consisting of four nucleoside triphosphates, where a fourth nucleoside triphosphate of the four nucleoside triphosphates is present in a rate-limiting amount;   detecting a pause in the movement of the target nucleic acid molecule and/or one or more nascent strand(s) through, on, or over the nanopore due to a pause in translocation of the target nucleic acid molecule and/or one or more nascent strand(s) by the polymerase;
 determining positional information of the first, second, third, and fourth nucleoside triphosphates along the target nucleic acid molecule based on the pause(s) in the movement of the target nucleic acid molecule and/or one or more nascent strand(s) through, on, or over the nanopore.