BIOCHEMICALLY ACTIVATED ELECTRONIC DEVICE

• US 20160017416A1
• Filed: 07/14/2015
• Published: 01/21/2016
• Est. Priority Date: 07/15/2014
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: BOYANOV, Boyan, MANDELL, Jeffrey G., GUNDERSON, Kevin L., CHEN, Cheng-Yao, Perbost, Michel, BAI, Jingwei
• Granted Patent: US 10,605,766 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 27/4145 specially adapted for biomolecules, e.g. gate electrode with immobilised receptors

Abstract

A method of nucleic acid sequencing. The method can include the steps of (a) providing a polymerase tethered to a solid support charge sensor; (b) providing one or more nucleotides, whereby the presence of the nucleotide can be detected by the charge sensor; and (c) detecting incorporation of the nucleotide into a nascent strand complementary to a template nucleic acid.

Claims

1. A method of nucleic acid sequencing comprising:

(a) providing a polymerase tethered to a solid support charge sensor;

(b) providing one or more labeled nucleotides, whereby the presence of the label can be detected by the charge sensor when the label is in proximity to the charge sensor; and

(c) detecting incorporation of the labeled nucleotide into a nascent strand complementary to a template nucleic acid using the charge sensor.

2. The method of claim 1, wherein the label comprises an oligonucleotide.

3. The method of claim 2, wherein the polymerase is tethered to the solid support charge sensor with a tether comprising nucleic acid and wherein the oligonucleotide is capable of hybridizing to the nucleic acid of the tether.

4. The method of claim 1, wherein the label is cleaved from the nucleotide by the polymerase after incorporation.

5. The method of claim 1, wherein the one or more types of labeled nucleotides comprise a plurality of different types of charge labels, and wherein the one or more labeled nucleotides comprise a unique charge label for each of four types of nucleotides.

6. The method of claim 1, wherein the charge sensor comprises a nanowire FET or a carbon nanotube.

7. The method of claim 1, wherein the solid support comprises a plurality of charge sensors that are tethered to polymerases, and step (c) comprises detecting incorporation of the labeled nucleotide into a nascent strand complementary to a template nucleic acid at each polymerase in the plurality of polymerases.

8. The method of claim 7, wherein the plurality of polymerases comprises at least two different types of polymerases, and wherein the different types of polymerases produce mutually distinguishable signals detectable by the charge sensors when incorporating the same type of nucleotide into a nascent nucleic acid strand.

9. The method of claim 8, wherein the detecting comprises distinguishing the mutually distinguishable signals and thereby determining the sequences for a plurality of nucleic acids.

10. The method of claim 1, wherein the polymerase is tethered to the solid support charge sensor with a conducting tether.

11. The method claim 1, wherein the polymerase lacks a 3′

to 5′

exonuclease function.

12. The method of claim 11, wherein the polymerase comprises the large fragment of Bsu DNA polymerase I.

13. The method of claim 12, wherein the large fragment of Bsu DNA polymerase I is modified to include residues from the O-helix finger domain of Klenow fragment.

14. The method of claim 1, wherein the solid support charge sensor comprises a first width within which is a region of substantially narrower width.

15. The method of claim 14, wherein the polymerase is tethered to the solid support charge sensor by attachment to the region of substantially narrower width.

16. The method of claim 1, wherein the polymerase is tethered to the solid support charge sensor with a tether comprising native ribonucleotides or analogs thereof.

17. The method of claim 16, wherein the label comprises native ribonucleotides or analogs thereof, and wherein the native ribonucleotides or analogs thereof of the label hybridize to the native ribonucleotides or analogs thereof of the tether.

18. The method of claim 16, wherein the label comprises native deoxyribonucleotides or analogs thereof, and wherein the native deoxyribonucleotides or analogs thereof of the label hybridize to the native ribonucleotides or analogs thereof of the tether.

19. The method of claim 1, wherein the polymerase is tethered to the solid support charge sensor with a tether comprising native deoxyribonucleotides or analogs thereof.

20. The method of claim 19, wherein the label comprises native ribonucleotides or analogs thereof, and wherein the native ribonucleotides or analogs thereof of the label hybridize to the native deoxyribonucleotides or analogs thereof of the tether.

21. A method of nucleic acid sequencing comprising:

(a) providing a polymerase tethered to a solid support charge sensor;

(b) contacting the polymerase with a template nucleic acid and one or more different nucleotide types under conditions wherein the polymerase catalyzes addition of the one or more nucleotide types to form a nucleic acid complement of the nucleic acid template, and wherein the addition of one or more different nucleotide types produces a conformational signal change from the polymerase that is detected by the charge sensor;

(c) detecting a change in the signal from the polymerase using the charge sensor; and

(d) determining the rate, polarity, amplitude or time duration for the change in the signal for the addition of the one or more different nucleotide type, thereby determining a sequence of nucleotides for the template nucleic acid.

22. A method of nucleic acid sequencing comprising:

(a) providing a polymerase tethered to a solid support charge sensor;

(b) providing one or more labeled nucleotides, whereby the presence of the label can be detected by the charge sensor when the label is in proximity to the charge sensor, wherein the one or more labeled nucleotides comprise reversible terminator moieties;

(c) detecting incorporation of the one or more labeled nucleotides into a nascent strand complementary to a template nucleic acid using the charge sensor, thereby forming a reversibly terminated nascent strand;

(d) modifying the reversible terminated nascent strand to render the nascent strand capable of further incorporation of nucleotide; and

(e) repeating steps (b) through (d) to obtain a sequence of the template nucleic acid.

23. A method for attaching reaction components to charge sensors, comprising

(a) providing a solid support comprising a plurality of charge sensors, wherein each of the charge sensors has a capacity to attach a plurality of reaction components;

(b) providing a fluid comprising a plurality of reaction components of a particular type, wherein each of the reaction components of the particular type is bound to repellant moiety; and

(c) contacting the solid support with the fluid under conditions wherein

(i) the plurality of reaction components of the particular type are in fluid communication with the plurality of charge sensors,

(ii) a greater number of reaction components of the particular type is in the fluid than the number of charge sensors on the solid support;

(iii) reaction components of the particular type from the fluid attach to the charge sensors, and

(iv) the repellant moiety bound to each of the reaction components prevents more than one of the reaction components in the plurality of reaction components from attaching to each of the charge sensors.

24. A method for attaching reaction components to charge sensors, comprising

(a) providing a solid support comprising a plurality of charge sensors,

wherein each of the charge sensors has a capacity to attach a plurality of reaction components;

(b) providing a fluid comprising a plurality of reaction components of a particular type;

(c) contacting the solid support with the fluid under conditions wherein

(i) the plurality of reaction components of the particular type are in fluid communication with the plurality of charge sensors,

(ii) a greater number of reaction components of the particular type is in the fluid than the number of charge sensors on the solid support; and

(iii) reaction components of the particular type from the fluid attach to the charge sensors, thereby forming modified charge sensors that are attached to multiple reaction components of the particular type from the fluid; and

(d) removing one or more of the reaction components of the particular type from each of the modified charge sensors to leave a single one of the reaction components of the particular type attached to each of the modified charge sensors.