SYSTEMS AND METHODS FOR NUCLEIC ACID SEQUENCING

• US 20180100190A1
• Filed: 07/20/2017
• Published: 04/12/2018
• Est. Priority Date: 07/20/2016
• Status: Active Grant

• Current Assignee: Sequencing Health Incorporated
• Original Assignee: GenapSys, Inc.
• Inventors: Esfandyarpour, Hesaam, Lau, Aldrich N.K., Parizi, Kosar, Paliwal, Saurabh, Persson, Henrik, Stern, Seth, Kenney, Paul, Barmi, Meysam R., Nabi, Ali, Rategh, Hamid, Oberstrass, Florian
• Granted Patent: US 10,544,456 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

Provided herein are systems and methods for processing and analyzing nucleic acids and other biomolecules. Methods may include processing nucleic acid molecules in an emulsion of droplets. Methods of analyzing nucleic acid molecules may include coupling nucleic acids to a bead or other support. Methods may include analysis of nucleic acid molecules using a redox mediator. In some cases, analysis of the nucleic acid molecule includes determining a nucleotide sequence of the nucleic acid molecule.

Claims

1. A method for sequencing a nucleic acid template, comprising:

(a) contacting a nucleic acid template with a sensing fluid containing a population of nucleotides, wherein said nucleic acid template is hybridized to a primer that is coupled to a bead, which bead is positioned proximate to a sensor in a sensor array, wherein said sensor comprises at least two electrodes, and wherein said sensing fluid has a bulk conductivity and a surface of said bead has a surface conductivity to provide a Dukhin number that is less than about 1;

(b) using said sensor to detect a change in conductivity within a Debye layer of said bead upon incorporation of at least one nucleotide of said population of nucleotides into a growing nucleic acid strand, which growing nucleic acid strand is derived from said primer and is complementary to said nucleic acid template;

(c) washing said sensor array to remove unincorporated nucleotides of said population of nucleotides from said sensor array; and

(d) repeating (a)-(c) to obtain sequence information for said nucleic acid template.

2. The method of claim 1, wherein a given electrode of said at least two electrodes is exposed to said sensing fluid.

3. The method of claim 1, wherein (b) further comprises detecting a change in impedance within said Debye layer of said bead upon incorporation of said at least one nucleotide.

4. The method of claim 3, wherein said change in impedance within said Debye layer is detected at steady state.

5. The method of claim 1, wherein said at least two electrodes are positioned within said Debye layer of said bead.

6. The method of claim 1, wherein said sensing fluid has a solute concentration between about 0.15 millimolar and about 6 millimolar.

7. The method of claim 1, further comprising, prior to (b):

(i) contacting said sensor array with a probe fluid, wherein said probe fluid has a bulk conductivity that is at least about 50 times greater than or 50 times less than said conductivity associated with said surface of said bead; and

(ii) using said sensor to detect signals that are indicative of a presence of said bead in proximity to said sensor.

8. The method of claim 7, wherein a Dukhin number determined from said bulk conductivity of said probe fluid and said conductivity of said surface of said bead is substantially less than 1.

9. The method of claim 7, wherein a Dukhin number determined from said bulk conductivity of said probe fluid and said conductivity of said surface of said bead is substantially greater than 1.

10. The method of claim 7, wherein (b), (c), and (d) are performed only at sensors of said sensor array at which signals indicative of bead occupancy are observed.

11. A method for determining bead occupancy at sites of a sensor array, comprising:

(a) contacting a sensor array with a plurality of beads, wherein said sensor array comprises a plurality of sensors each having at least two electrodes, to provide a given bead of said plurality of beads at a given position in proximity to an individual sensor of said plurality of sensors;

(b) contacting said sensor array with a probe fluid that has a bulk conductivity that is at least about 50 times greater than or 50 times less than a conductivity associated with a surface of said given bead;

(c) using said individual sensor to detect signals that are indicative of a presence of said given bead in proximity to said sensor; and

(d) identifying said given position of said sensor array as occupied by said given bead.

12. The method of claim 11, wherein said probe fluid has a concentration of solutes between about 0.01 millimolar and about 1 molar.

13. The method of claim 11, wherein a Dukhin number determined from said bulk conductivity of said probe fluid and said conductivity of said surface of said bead is substantially less than 1.

14. The method of claim 11, wherein a Dukhin number determined from said bulk conductivity of said probe fluid and said conductivity of said surface of said bead is substantially greater than 1.

15. The method of claim 11, further comprising a nucleic acid coupled to said given bead.

16. The method of claim 11, wherein said signals comprise electrical current.

17. The method of claim 11, wherein a given electrode of said individual sensor is positioned within a Debye layer of said given bead.

18-31. -31. (canceled)

32. A method for sequencing a nucleic acid molecule, comprising:

a. activating a sensor comprising a support comprising at least two electrodes and a polymeric material adjacent to said support, wherein said at least two electrodes are exposed to a solution comprising said polymeric material, wherein said polymeric material retains said nucleic acid molecule during a sequencing reaction;

b. subjecting said nucleic acid molecule to said sequencing reaction to yield signals indicative of individual bases of said nucleic acid molecule;

c. during said sequencing reaction, using said at least two electrodes of said sensor to detect said signals; and

d. using said signals detected in (c) to generate a sequence of said nucleic acid molecule.

37. The method of claim 32, wherein said polymeric material is a hydrogel.

45. The method of claim 32, wherein during said sequencing reaction, said at least two electrodes are coupled to a Debye layer having of said nucleic acid molecule.

33-36. -36. (canceled)

38-44. -44. (canceled)

46-79. -79. (canceled)