Characterization of individual polymer molecules based on monomer-interface interactions

Patent metadata

Field	Value
Publication number	US-8986528-B2
Application number	US-201313852606-A
Country	US
Kind code	B2
Filing date	Mar 28, 2013
Priority date	Mar 17, 1995
Publication date	Mar 24, 2015
Grant date	Mar 24, 2015

Abstract

The invention relates to a method for characterizing a target polynucleic acid by providing a surface containing a channel of a dimension sufficient to allow sequential monomer-by-monomer passage of a single-stranded polynucleic acid, but not of a double-stranded polynucleic acid; providing a source of hybridized target polynucleic acid at the surface; inducing passage of the target polynucleic acid through the channel, whereby the target polynucleic acid undergoes base pair separation (melts) prior to its passage; and making one or more measurements over time as the target polynucleic acid moves relative to the channel yielding data suitable to determine a monomer-dependent characteristic of the target polynucleic acid.

First claim

What is claimed is:

1. A method for characterizing a target polynucleic acid comprising:
a) providing a surface containing a channel of a dimension sufficient to allow sequential monomer-by-monomer passage through the channel of a single-stranded polynucleic acid, but not of a double-stranded polynucleic acid;
b) providing a source of hybridized target polynucleic acid at the surface;
c) applying a voltage across the channel to drive the target polynucleic acid to the channel so that a single strand of the target polynucleic acid enters the channel and to hold the target polynucleic acid adjacent to the channel for a time sufficient for the hybridized target polynucleic acid to undergo strand separation at the surface, wherein the single strand of the target polynucleic acid passes through the channel; and
d) making one or more measurements over time as the single strand of the target polynucleic acid moves relative to the channel yielding data suitable to determine a polymer-dependent property of the target polynucleic acid.

2. A method according to claim 1 wherein the channel connects two separate pools of solution and comprising providing a source of hybridized target polynucleic acid in one of the two pools.

3. A method according to claim 1 comprising measuring current flow through the channel in the presence of a potential difference between the two pools.

4. A method according to claim 1 wherein the channel is an ion-permeable passage.

5. A method according to claim 4 wherein the channel is a protein pore.

6. A method according to claim 5 wherein the protein pore is a bacterial porin.

7. A method according to claim 5 wherein the protein pore is naturally occurring, recombinant or mutant.

8. A method according to claim 5 wherein the channel is voltage-sensitive.

9. A method according to claim 1 wherein the channel is synthetic.

10. A method according to claim 1 wherein the applied voltage is variable.

11. A method according to claim 1 wherein the surface comprises more than one channel of a dimension sufficient to allow sequential monomer-by-monomer passage of a single-stranded polynucleic acid, but not of a double-stranded polynucleic acid.

12. A method according to claim 1 wherein the hybridized target polynucleic acid comprises a hairpin loop.

13. A method according to claim 1 further comprising monitoring the duration or magnitude of a flow blockage in the channel, and, following detection of a flow blockage, reversing the potential across the channel to remove the flow blockage.

14. A method according to claim 1 , wherein step (d) comprises making one or more measurements over time as the single strand of the target polynucleic acid moves relative to the channel yielding data suitable to distinguish the target polynucleic acid from another polynucleic acid.

15. A method according to claim 1 , wherein step (d) comprises making one or more measurements over time as the single strand of the target polynucleic acid moves relative to the channel yielding data suitable to determine a sequence of the target polynucleic acid.

Assignees

• Harvard College

Inventors

• Denison Timothy J
• Sauer-Budge Alexis
• Golovchenko Jene A
• Meller Amit
• Brandin Eric
• Branton Daniel

Classifications

• G01N27/447 Primary

  using electrophoresis · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N33/48721

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q2565/607

  being a sensor, e.g. electrode · CPC title