Devices and methods for determining the length of biopolymers and distances between probes bound thereto

• US 9,719,980 B2
• Filed: 07/15/2014
• Issued: 08/01/2017
• Est. Priority Date: 09/03/2008
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: NABsys, Inc.
• Inventors: Oliver, John S.
• Primary Examiner(s): Van, Luan
• Assistant Examiner(s): ROSENWALD, STEVEN ERIC
• Application Number:
  US14/331,629
• Publication Number:
  US 20150008124A1
• Time in Patent Office: 1,113 Days
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 27/4473 by electric means
• CPC Class Codes: G01N 33/48721 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 Class Codes: Y10S 977/957 Of chemical property or presence

Abstract

Devices and methods for detecting the length of analytes and/or sequencing analytes are provided in which two or more electrical signals are obtained as an analyte traverses a fluidic channel. Detection of the relative position of probes hybridized to a biopolymer and/or the length of the analyte (e.g., a biopolymer) does not rely on the absolute time between detection events of a given electrical signal to determine a distance associated with the biopolymer. Instead, multiple signals are obtained (e.g., as functions of time) corresponding to a plurality of detector volumes at known locations along a fluidic channel through which the biopolymer passes, and the distances are determined from the multiple signals.

Claims

1. A method for sequencing a biopolymer, the method comprising:

preparing an analyte by hybridizing a first plurality of probes with the biopolymer such that said first plurality of probes attaches to portions of the biomolecule to produce a partially hybridized biomolecule;

disposing the analyte in a fluidic channel;

applying a potential along the fluidic channel;

translocating the analyte from a first end of the fluidic channel to a second end of the fluidic channel;

detecting two or more coincident electrical signals as the analyte moves through the fluidic channel, said two or more electrical signals corresponding to two or more detector volumes of the fluidic channel, said two or more electrical signals being detected by using a plurality of sensing electrodes disposed along the length of the fluidic channel while not directly applying voltage to the sensing electrodes with a voltage source, each detector volume being defined by two sensing electrodes laterally offset from each other along the fluidic channel, the detected electrical signals indicating locations of the hybridized probes along the biopolymer;

analyzing the coincident electrical signals to determine in which detector volumes probes bound to the biomolecule are located;

determining at least a portion of the sequence of the biopolymer using a distance between sensing electrodes corresponding to said detector volumes in which the probes are located,

wherein (i) said sensing electrodes are configured for connection to a measurement tool for capturing said electrical signals corresponding to said detector volumes, (ii) the fluidic channel is a nanochannel, (iii) relative positions of the sensing electrodes are known, and (iv) the captured electrical signals in conjunction with the relative positions of the sensing electrodes indicate at least the portion of the sequence of the biopolymer.

2. The method of claim 1 wherein applying the potential along the fluidic channel generates an electrophoretic force therein.

3. The method of claim 1, wherein translocating the analyte comprises using a chemical gradient.

4. The method of claim 1, wherein translocating the analyte comprises using a pressure differential.

5. The method of claim 1, further comprising determining a distance between probes using a coincident response of electrical signals corresponding to two or more detector volumes.

6. The method of claim 5, wherein a spacing between sensing electrodes is used to determine a maximum distance between probes.

7. The method of claim 5, wherein a spacing between sensing electrodes is used to determine a minimum distance between probes.

8. The method of claim 1, wherein each of the electrical signals initially changes when the biopolymer moves through a corresponding detector volume associated with two sensing electrodes and further changes when a portion of the biopolymer comprising a hybridized probe moves through the detector volume.

9. The method of claim 1, further comprising hybridizing a second plurality of probes with the biopolymer and repeating the detecting, analyzing, and determining steps with said second plurality of probes.

10. The method of claim 9, wherein a complexed region of the biopolymer comprises at least one hybridized probe and an uncomplexed region of the biopolymer is free of a hybridized probe, further comprising using the two or more electrical signals to detect and record complexed and uncomplexed regions of the biopolymer to create a first probe map of the first plurality of probes and a second probe map of the second plurality of probes, the first probe map and the second probe map respectively comprising information about the relative positions of the hybridized first and second plurality of probes.

11. The method of claim 10, further comprising determining a candidate sequence by ordering at least two probe sequences using at least one of positional information or a combination of overlapping probe binding sequences and positional information.

12. The method of claim 10, wherein the first and second probe maps further comprise information about an error of the positional information for each probe, wherein the error is an uncertainty of a distance between the probes of a hybridized plurality of probes.

13. The method of claim 12, further comprising determining a candidate sequence by ordering at least two probe sequences using at least one of (i) positional information and parameters relating to the error in positional information or (ii) a combination of overlapping sequences of the probe molecules and positional information and the error in positional information.

14. The method of claim 10, wherein the biopolymer comprises a double-stranded biopolymer target molecule.

15. The method of claim 14, wherein preparing the analyte comprises contacting the biopolymer with a first probe having a first probe specificity for recognition sites of the biopolymer to form a first plurality of local ternary complexes, the first probe having a first known recognition site sequence.

16. The method of claim 15, further comprising using the electrical signal to determine positional information of the first plurality of local ternary complexes.

17. The method of claim 15, wherein preparing the analyte further comprises contacting the biopolymer with a second probe having a second probe specificity for recognition sites of the biopolymer to form a second plurality of local ternary complexes, the second probe having a second known recognition site sequence.

18. The method of claim 17, further comprising aligning positional information of at least the first and second plurality of local ternary complexes to determine a sequence of the biopolymer.