Systems and methods for nucleic acid sequencing

• US 10,544,456 B2
• Filed: 07/20/2017
• Issued: 01/28/2020
• Est. Priority Date: 07/20/2016
• Status: Active Grant

• Current Assignee: Sequencing Health Incorporated
• Original Assignee: GenapSys, Inc.
• Inventors: Esfandyarpour, Hesaam, Parizi, Kosar, Paliwal, Saurabh, Stern, Seth, Kenney, Paul, Barmi, Meysam R., Nabi, Ali, Rategh, Hamid
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/655,616
• Publication Number:
  US 20180100190A1
• Time in Patent Office: 922 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, wherein said sensing fluid has a sensing fluid bulk conductivity and a surface of said bead has a surface conductivity to provide a Dukhin number that is less than 1 such that (i) a conductivity measurement by said at least two electrodes with said bead positioned proximate to said sensor is substantially similar to (ii) another conductivity measurement by said at least two electrodes without said bead positioned proximate to said sensor;

(b) using said at least two electrodes of said sensor to detect a conductivity change 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, which conductivity change is detected based at least in part on an electrical current change through said Debye layer;

(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 an 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 probe fluid bulk conductivity that is at least about 50 times greater than or at least about 50 times less than said surface conductivity of 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 an additional Dukhin number determined from said probe fluid bulk conductivity and said surface conductivity of said surface of said bead is substantially less than 1.

9. The method of claim 7, wherein an additional Dukhin number determined from said probe fluid bulk conductivity and said surface 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.