CHIP SET-UP AND HIGH-ACCURACY NUCLEIC ACID SEQUENCING

• US 20140034497A1
• Filed: 06/14/2013
• Published: 02/06/2014
• Est. Priority Date: 06/15/2012
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Genia Technologies, Inc. (Roche Holding AG)
• Inventors: Davis, Randall, Chen, Roger, Bibillo, Arkadiusz, Deierling, Kevin
• Granted Patent: US 9,494,554 B2
• Time in Patent Office: Days
• US Class Current: 204/451
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• 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

Abstract

The present disclosure provides devices, systems and methods for sequencing nucleic acid molecules. Nucleic acid molecules can be sequenced with a high accuracy (e.g., greater than 97% in a single pass) using a chip comprising an array of independently addressable nanopore sensors at a density of at least about 500 sites per 1 mm². An individual nanopore sensor can include a nanopore in a membrane that is adjacent or in proximity to a sensing electrode.

Claims

1. A method for nucleic acid sequencing, comprising:

(a) providing a chip comprising a plurality of discrete sites at a density greater than or equal to about 500 sites per 1 mm², wherein an individual site of said plurality of discrete sites comprises at least one nanopore formed in a membrane disposed adjacent to an electrode, wherein each discrete site is adapted to aid in the detection of said nucleic acid molecule or a portion thereof;

(b) directing a plurality of nucleic acid molecules to said plurality of discrete sites; and

(c) characterizing, with the aid of a computer processor coupled to said discrete sites, a nucleic acid sequence of each of said nucleic molecules based on electrical signals received from said plurality of discrete sites.

2. The method of claim 1, wherein said plurality of nucleic acid molecules are derived from a nucleic acid sample.

3. The method of claim 2, wherein each of said plurality of nucleic acid molecules has a shorter nucleic acid sequence than said nucleic acid sample.

4. The method of claim 3, further comprising, prior to (b), fragmenting said nucleic acid sample to provide said plurality of nucleic acid molecules.

5. The method of claim 2, further comprising characterizing a nucleic acid sequence of said nucleic acid sample based upon a characterization of a nucleic acid sequence of each of said nucleic molecules.

14. The method of claim 1, wherein each discrete site is adapted to regulate molecular flow through or adjacent to said at least one nanopore.

15. The method of claim 1, wherein each discrete site is adapted to detect said nucleic acid molecule or a portion thereof upon molecular flow of said nucleic acid molecule or portion thereof through or adjacent to said at least one nanopore.

16. The method of claim 1, wherein said nucleic acid sequence is characterized upon movement of each of said plurality of nucleic acid molecules or portions thereof.

17. The method of claim 1, wherein said electrode is coupled to an integrated circuit that processes a signal detected with the aid of said electrode.

18. The method of claim 1, wherein said computer processor is external to the chip.

19. The method of claim 1, wherein each discrete site is independently addressable.

6-13. -13. (canceled)

20. A system for sequencing a nucleic acid molecule, comprising:

(a) a chip comprising a plurality of discrete sites at a density greater than or equal to about 500 sites per 1 mm², wherein an individual site of said plurality of discrete sites comprises at least one nanopore formed in a membrane disposed adjacent to an electrode, wherein each discrete site is adapted to aid in the detection of said nucleic acid molecule or a portion thereof; and

(b) a processor coupled to said chip, wherein said processor is programmed to aid in characterizing a nucleic acid sequence of said nucleic acid molecule based on electrical signals received from said plurality of discrete sites.

21-45. -45. (canceled)

46. A device for use in molecular sensing comprising one or more nanopores in a membrane disposed upon at least one membrane incompatible surface in sensing proximity to an electrode coupled to an integrated circuit, wherein said membrane comprising said one or more nanopores exhibits a capacitance greater than about 5 fF/μ

m² and a conductance less than about 10 nano Siemens per mole of electrolyte per nanopore as measured by said electrode under an applied voltage of at least about 50 mV.

47. The device of claim 46, wherein said integrated circuit is an individually addressable integrated circuit.

48. The device of claim 46, wherein said integrated circuit comprises a logic controller.

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

(a) providing a chip comprising an array of sensors, wherein an individual sensor of said array of sensors comprises a membrane adjacent to a sensing electrode, wherein said membrane comprises at least one nanopore configured to aid in the detection of one or more nucleic acid bases of said nucleic acid molecule or portion thereof upon the flow of said nucleic acid molecule or portion thereof through or adjacent to said at least one nanopore;

(b) directing said nucleic acid molecule to said individual sensor;

(c) applying a series of electrical pulses to said membrane upon directing said nucleic acid molecule to said individual sensor; and

(d) detecting one or more nucleic acid bases of said nucleic acid molecule or portion thereof between said series of electrical pulses.

50-57. -57. (canceled)

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

(a) providing a chip comprising an array of individual sensors, wherein an individual sensor of said array comprises an electrode that is disposed adjacent to a membrane having a nanopore therein, wherein said electrode is coupled to an electrical circuit that is adapted to generate an electrical signal to aid in the detection of said nucleic acid molecule or a portion thereof upon the flow of said nucleic acid molecule or portion thereof through or in proximity to said nanopore;

(b) directing said nucleic acid molecule or portion thereof through or in proximity to said nanopore; and

(c) identifying a nucleic acid sequence of said nucleic acid molecule or portion thereof at an accuracy of at least about 97%.

59. The method of claim 58, wherein said nucleic acid sequence is generated with the aid of a computer processor coupled to said electrical circuit.

62. The method of claim 58, wherein said individual sensor is independently addressable.

63. The method of claim 58, wherein said nucleic acid molecule comprises a tag that is detected by said nanopore upon the incorporation of complementary nucleic acid bases into said nucleic acid molecule.

73. The method of claim 58, wherein said nucleic acid sequence of said nucleic acid molecule or portion thereof is identified by combining data collected from at least 10 passes of said nucleic acid molecule or portion thereof through or in proximity to said nanopore.

74. The method of claim 73, wherein said nucleic acid sequence of said nucleic acid molecule or portion thereof is identified by combining data collected from at least 20 passes of said nucleic acid molecule or portion thereof through or in proximity to said nanopore.

60-61. -61. (canceled)

64-72. -72. (canceled)

75. A system for sequencing a nucleic acid molecule, comprising:

(a) a chip comprising an array of individual sensors, wherein an individual sensor of said array comprises an electrode that is disposed adjacent to a membrane having a nanopore therein, wherein said electrode is coupled to an electrical circuit that is adapted to generate an electrical signal to aid in the detection of said nucleic acid molecule or a portion thereof upon the flow of said nucleic acid molecule or portion thereof through or adjacent to said nanopore; and

(b) a processor coupled to said chip, wherein said processor is programmed to aid in characterizing a nucleic acid sequence of said nucleic acid molecule based on electrical signals received from said plurality of discrete sites at an accuracy of at least about 97%.

76-87. -87. (canceled)

88. A method for sequencing a nucleic acid sample, comprising:

a) facilitating, without the use of a molecular motor, the flow of said nucleic acid sample through a nanopore embedded in a membrane;

b) detecting one or more nucleic acid subunits of said nucleic acid sample upon the flow of said nucleic sample through said nanopore;

c) sequencing said nucleic acid sample upon detecting said one or more nucleic acid subunits.

89-98. -98. (canceled)

99. A system for sensing a biological sample from a subject, the system comprising:

a) a housing;

b) a sensor within the housing, the sensor having an electrical circuit adjacent to a membrane with a nanopore therein, wherein the electrical circuit is adapted to generate an electrical signal in response to the biological sample flowing through or adjacent to the nanopore; and

c) an identification member on or within the housing, the identification member having a unique identifier associated with the system and adapted to aid in associating the electrical signal, or characteristic information derived from the electrical signal, with the subject.

100-102. -102. (canceled)

103. A method for manipulating a fluid on a surface, comprising:

(a) providing a surface, an array of electrodes in proximity to the surface, and a fluid comprising a hydrophilic phase and a hydrophobic phase, wherein said hydrophobic phase is adjacent to said surface; and

(b) energizing the electrodes in a spatial and/or temporal pattern, thereby decreasing a volume of said hydrophobic phase in relation to said hydrophilic phase in proximity to said electrodes.

104-116. -116. (canceled)

117. A biochip, comprising a nanopore in a membrane that is disposed within, adjacent to, or in proximity to a well, wherein the well comprises an electrode that is capable of detecting a change in the flow of ions through said nanopore in response to a species passing through, in proximity to, or adjacent to the nanopore, wherein said electrode is capable of detecting said change in the flow of ions for at least 1 hour without re-adjusting the ion concentrations on either side of the membrane.

118-120. -120. (canceled)

121. A method for forming a biochip, the method comprising:

(a) providing a semiconductor substrate;

(b) forming a plurality of wells in said semiconductor substrate at a density of at least 500 wells/mm²;

(c) forming an electrode in an individual well of said plurality, wherein said electrode is capable of performing electrical measurements of detectable species that are disposed on or adjacent to the semiconductor substrate, and wherein said electrode has an operating life of at least 15 minutes with 40 mV applied potential; and

(d) preparing the substrate for the formation of a membrane that seals the individual well at a resistivity of at least about 10 gigaohms.

122-125. -125. (canceled)