Real-time electronic sequencing

• US 9,708,656 B2
• Filed: 05/05/2014
• Issued: 07/18/2017
• Est. Priority Date: 05/06/2013
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Hanes, Jeremiah, Turner, Stephen, Bjornson, Keith
• Primary Examiner(s): Forman, Betty
• Application Number:
  US14/270,099
• Publication Number:
  US 20150065353A1
• Time in Patent Office: 1,170 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• 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 2563/157 Nanotubes or nanorods
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 27/227 Sensors changing capacitance upon adsorption or absorption of fluid components, e.g. electrolyte-insulator-semiconductor sensors, MOS capacitors G01N27/225 takes precedence
• CPC Class Codes: G01N 27/4145 specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
• CPC Class Codes: G01N 27/4146 involving nanosized elements, e.g. nanotubes, nanowires

Abstract

Real time electronic sequencing methods, devices, and systems are described. Arrays of nanoscale electronic elements comprising capacitive devices with one or two electrodes, or arrays of nanoFET devices are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound proximate to the nanoscale electronic elements or to the substrate proximate to the nanoscale electronic element. A sequencing reaction mixture comprising nucleotide analogs having impedance labels, capacitive labels, or conductivity labels is introduced to the array of nanoscale electronic elements comprising capacitive devices or nanoFETs under conditions of polymerase mediated nucleic acid synthesis. The time sequence of incorporation of nucleotide analogs is determined by identifying the types of labels of the nucleotide analogs that are incorporated into the growing strand using measured impedance, conductivity, or capacitance.

Claims

1. A method for nucleic acid sequencing comprising:

providing a substrate comprising an array of nanoFETs, each comprising a source, a drain, and a gate, wherein a plurality of the nanoFETs comprise a single polymerase enzyme complex comprising a polymerase enzyme and a template nucleic acid, the complex attached to gate of the nanoFET or to the substrate proximate to the gate of the nanoFET;

providing a plurality of types of nucleotide analogs, each type of nucleotide comprising a conductivity label attached to the phosphate portion of the nucleotide analog through a linker, wherein each type of nucleotide has a different linker, and each different linker is selected such that each type of nucleotide exhibits different current oscillation frequency characteristics;

exposing the substrate to the plurality of types of nucleotide analogs under conditions whereby polymerase mediated nucleic acid synthesis occurs, resulting in cleavage of the conductivity label and the growth of a nascent nucleic acid strand;

applying a voltage between the source and drain, whereby when a nucleotide analog resides in the active site of the enzyme, the conductivity label on the nucleotide analog produces a measurable change in the conductivity of the gate;

monitoring an electrical signal at the nanoFET over time to detect an incorporation event for a nucleotide analog, the electrical signal including current oscillation frequency while the nucleotide analog is associated with the polymerase enzyme using the monitored electrical signal to identify the type of nucleotide incorporated; and

using a plurality of monitored electrical signals to determine a sequence of the template nucleic acid.

2. The method of claim 1 wherein the electrical signal used to determine the sequence of the template nucleic acids further includes the duration of the signal indicating the residence time of a nucleotide analog in the active site of a polymerase.

3. The method of claim 1 wherein the gate of each nanoFET comprises a nanowire.

4. The method of claim 1 wherein the gate of each nanoFET comprises doped silicon.

5. The method of claim 1 wherein the voltage across the source and drain is DC.

6. The method of claim 1 wherein the voltage across the source and drain is AC, and the frequency of the AC voltage is changed with time.

7. The method of claim 1 wherein the plurality of types of nucleotide analogs comprise four types of nucleotide analogs corresponding to A, G, C, T, or A, G, C, U.

8. The method of claim 7 wherein the conductivity labels comprise nanoparticles.

9. The method of claim 8 wherein the nanoparticles have sizes from about 2 nm to about 50 nm.

10. The method of claim 7 wherein some of the nucleotide types have different conductivity label types.

11. The method of claim 10 wherein the nucleotide types comprise at least one positively charged conductivity label and at least one negatively charged conductivity label.

12. The method of claim 1 wherein the gate of the nanoFET comprises a carbon nanotube.

13. The method of claim 1 wherein the gate of the nanoFET comprises a single-walled carbon nanotube.

14. The method of claim 1 wherein the substrate is exposed to four types of nucleotide analogs corresponding to A, G, C, T, or A, G, C, U, each of the four types of nucleotide analogs having a different length linker.

15. The method of claim 1 wherein at least one of the linkers comprises polyethylene glycol.