Single molecule nanoFET sequencing systems and methods

• US 10,125,391 B2
• Filed: 08/03/2016
• Issued: 11/13/2018
• Est. Priority Date: 08/06/2015
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Korlach, Jonas, Kamtekar, Satwik, Hanes, Jeremiah
• Primary Examiner(s): Forman, Betty J
• Application Number:
  US15/227,661
• Publication Number:
  US 20170037462A1
• Time in Patent Office: 832 Days
• Field of Search: None
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• 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 1/6869 Methods for sequencing
• 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 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• 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
• CPC Class Codes: G01N 27/4148 Integrated circuits therefor, e.g. fabricated by CMOS processing CMOS processing per se H01L21/82
• CPC Class Codes: G11C 19/28 using semiconductor elements G11C19/14, G11C19/36 take precedence
• CPC Class Codes: H01L 29/0669 Nanowires or nanotubes carbon nanotubes as material of solid-state device active part H10K85/211
• CPC Class Codes: H01L 29/0673 oriented parallel to a substrate
• CPC Class Codes: H01L 29/1606 Graphene

Abstract

Real time electronic sequencing methods, devices, and systems are described. Arrays of nanoFET devices are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound to the nanoFET. A sequencing reaction mixture comprising nucleotide analogs having conductivity labels is introduced to the array of nanoscale electronic elements comprising nanoFETs under conditions of polymerase mediated nucleic acid synthesis. The polymerase enzyme template complex is attached to the gate of the nanoFET in an orientation whereby the nucleotide exit region of the polymerase enzyme is directed toward the gate of the nanoFET. Methods for producing nanoFET arrays are provided.

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 the gate of the nanoFET, wherein the polymerase enzyme is attached to the gate in an orientation whereby the nucleotide exit region of the polymerase enzyme is toward the gate of the nanoFET, wherein the polymerase comprises a phi29 type polymerase, wherein the polymerase is attached through two linkers at two different positions on the polymerase, and wherein at least one of the two linkers is attached to the polymerase within 5 amino acids from position 375 or within 5 amino acids from position 512;

exposing the substrate to a plurality of types of nucleotide analogs, each comprising a different conductivity label attached to the phosphate portion of the nucleotide analog through a linker 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 electrical signal at the gate;

monitoring an electrical signal at the gate over time, whereby the electrical signal indicates an incorporation event for a type of nucleotide analog having a specific conductivity label; and

using the electrical signal 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 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 a carbon nanotube.

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 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 conductivity label.

8. The method of claim 1 wherein the conductivity label comprises a protein.

9. The method of claim 8 wherein the protein has a molecular weight that is between 1/10and 3 times the molecular weight of the polymerase enzyme.

10. The method of claim 8 wherein the protein has a molecular weight that is between 1/10and 3 times the molecular weight of a phi29 polymerase.

11. The method of claim 1 wherein at least one of the conductivity labels comprises a polymer chain having multiple charges.

12. The method of claim 1 wherein there are 4 types of nucleotide analogs and each comprises a conductivity label comprising a polymer chain having multiple charges.

13. The method of claim 1 wherein there are 4 types of nucleotide analogs and each comprises a conductivity label having a different number of negative charges.

14. The method of claim 1 wherein there are 4 types of nucleotide analogs and each comprises a conductivity label having a different number of positive charges.

15. The method of claim 1 wherein there are 4 types of nucleotide analogs and each comprises a conductivity label having both negative and positive charges and each has a different net charge.

16. The method of claim 1 wherein there are 4 types of nucleotide analogs and two labels have a net negative charge, and two labels have a net positive charge.

17. The method of claim 1 wherein there are 4 types of nucleotide analogs and two of the labels result in an increase in conductivity at the gate when their corresponding nucleotide analog is associated with the polymerase, and two of the labels result in an decrease in conductivity at the gate when their corresponding nucleotide analog is associated with the polymerase.

18. The method of claim 1 wherein the phi29 type polymerase is a phi29 polymerase.

19. The method of claim 1 wherein the phi29 type polymerase is a B103, GA-1, PZA, Φ

15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, or Φ

21 polymerase.