Compositions, devices, systems, and methods for using a nanopore

a nanopore and nanopore technology, applied in the field of nanopore analysis techniques, can solve the problems of difficult correlation between the length of any given polynucleotide and its translocation time, and the control of the rate at which the target polynucleotide is analyzed in the nanopore analysis technique, so as to maintain enzyme activity

Inactive Publication Date: 2011-01-13
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0187]In one embodiment, the invention may be used to perform sequence analysis of polynucleotides. The analyses have an advantage over the prior art and the current art in that a single analysis may be performed at a single site, thereby resulting in considerable cost savings for reagents, substrates, reporter molecules, and the like. Of additional import is the rapidity of the sequencing reaction and the signal generated, thereby resulting in an improvement over the prior art.

Problems solved by technology

One disadvantage of previous nanopore analysis techniques is controlling the rate at which the target polynucleotide is analyzed.
Therefore, the correlation between any given polynucleotide's length and its translocation time is not straightforward.

Method used

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  • Compositions, devices, systems, and methods for using a nanopore
  • Compositions, devices, systems, and methods for using a nanopore
  • Compositions, devices, systems, and methods for using a nanopore

Examples

Experimental program
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examples

[0248]Herein are described several examples to demonstrate the capability of measuring macromolecules and polanions or polycations.

example i

Enzyme Binding is Prevented by a Blocking Primer

[0249]For an illustration of this method, see FIGS. 1(a) through 1(g). (a) In this scenario, the blocking primer is bound to the primer / template in bulk phase. Structure of the ternary complex prevents binding of the enzyme to the junction between the dsDNA and ssDNA segments of the target DNA where the first nucleotide would be incorporated. (b) Capture of a blocked primer / template under an applied voltage (trans side positive) threads the ssDNA into the pore and perches the dsDNA above the vestibule. This occurs because the loop at the end of the blocking primer is too large to enter the vestibule. The current reports capture of the complex in this state. (c) Under the applied voltage, the ssDNA segment advances in the pore toward the trans-side and processively unzips base-pairs between the blocking primer and the template. The energy cost of releasing each base pair independently is small (about 2.5 kcal / mol), so it proceeds rapidl...

example ii

Enzyme Catalysis is Prevented by a Blocking Primer

[0250]For an illustration of this method, see FIGS. 2(a) through 2(g). (a) In this scenario, the blocking primer is bound to the primer / template in bulk phase. Structure of the ternary complex permits binding of the enzyme to the target DNA but catalysis and processing along the template are prevented. (b) Capture of a blocked primer / template under an applied voltage (trans-side positive) threads the ssDNA into the pore and perches the dsDNA above the vestibule. This occurs because the loop at the end of the blocking primer is too large to enter the vestibule. The current reports capture of the complex in this state. (c) Under the applied voltage, the ssDNA segment advances in the pore toward the trans-side and processively unzips base-pairs between the blocking primer and the template. The energy cost of releasing each base pair independently is small (about 2.5 kcal / mol), so it proceeds rapidly under force. During this unzipping pr...

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Abstract

The invention herein disclosed provides for devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore. The devices and methods are also used to determine rapidly (˜>50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore. The invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.

Description

[0001]The present application claims priority to and benefits of the following: U.S. Provisional Patent Application Ser. No. 60 / 921,787 entitled “Methods To Limit Enzyme Activity To One Molecule Or Complex Using A Nanopore”, filed 4 Apr. 2007, U.S. Provisional Patent Application Ser. No. 60 / 931,115 entitled “Methods For Sequencing Polynucleotides By Synthesis Using A Nanopore”, filed 21 May, 2007, U.S. Provisional Patent Application Ser. No. 60 / 962,530 entitled “Methods For Positioning Single Molecules At A Defined Site” filed 30 Jul. 2007, U.S. Provisional Patent Application Ser. No. 60 / 967,539 entitled “Methods For Manufacture Of Very Large Scale Arrays Of Independently Addressable Nanopores And Methods For Their Use”, filed 4 Sep. 2007, and U.S. Provisional Patent Application Ser. No. 61 / 062,391 entitled “Feedback Control Of A Single Tethered Polynucleotide Suspended In A Nanopore To Repeatedly Probe Polynucleotide-Binding Proteins”, filed 25 Jan. 2008, all of which are herein in...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N27/26G01N27/40C25B3/29
CPCC12Q1/6869C25B3/10G01N33/48721C25B3/29C12Q2522/101C12Q2565/631C12Q1/25G01N33/573C12Q1/54C12Q1/6874G01N27/3278G01N27/4166C12Q2525/301C12Q2523/307C12Q2521/101C12Q2527/127
Inventor AKESON, MARK A.DEAMER, DAVID W.CHEN, ROGER JINTEH ARRIGOBENNER, SEICODUNBAR, WILIAM B.WILSON, NOAH A.LIEBERMAN, KATEABU-SHUMAYS, ROBINHURT, NICHOLAS
Owner RGT UNIV OF CALIFORNIA
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