Methods and systems for electronic sequencing

• US 8,969,002 B2
• Filed: 10/01/2012
• Issued: 03/03/2015
• Est. Priority Date: 10/04/2010
• Status: Active Grant

• Current Assignee: Sequencing Health Incorporated
• Original Assignee: GenapSys, Inc.
• Inventors: Esfandyarpour, Hesaam, Oldham, Mark
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Zhang, Kaijiang
• Application Number:
  US13/632,513
• Publication Number:
  US 20130096013A1
• Time in Patent Office: 883 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B03C 1/286 : disposed at the inner circumference of a recipient, e.g. magnetic drain bolt
• CPC Class Codes: B03C 1/288 : disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 1/30 : Combinations with other devices, not otherwise provided for
• CPC Class Codes: B03C 2201/26 : for use in medical applications
• CPC Class Codes: B03C 5/005 : Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength B03C5/02 takes precedence; electrophoresis B01D57/02
• CPC Class Codes: B03C 5/022 : Non-uniform field separators
• CPC Class Codes: B03C 5/026 : using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• CPC Class Codes: C12Q 1/6848 : characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 : using modified primers or templates
• 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 2523/307 : Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2563/116 : electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2563/143 : Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2565/543 : characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C40B 60/10 : for identifying library members
• CPC Class Codes: G01N 1/40 : Concentrating samples
• CPC Class Codes: G01N 2001/4038 : electric methods, e.g. electromigration, electrophoresis, ionisation
• 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 33/5438 : Electrodes
• CPC Class Codes: H01L 29/00 : Semiconductor devices specially adapted for rectifying, amplifying, oscillating or switching and having potential barriers; Capacitors or resistors having potential barriers, e.g. a PN-junction depletion layer or carrier concentration layer; Details of semiconductor bodies or of electrodes thereof ; Multistep manufacturing processes therefor H01L31/00 - H01L33/00, H10K10/00, H10N take precedence; details other than of semiconductor bodies or of electrodes thereof H01L23/00; devices consisting of a plurality of solid state components formed in or on a common substrate H01L27/00

Abstract

The present invention provides for methods and systems for Electronic DNA sequencing, single molecule DNA sequencing, and combinations of the above, providing low cost and convenient sequencing.

Claims

1. A method for sequencing a nucleic acid template, comprising:

a) contacting the nucleic acid template hybridized to a primer and coupled to a particle, which particle is proximate to a nanosensor in a nanosensor array, with a fluid containing nucleotides, wherein said nanosensor comprises a pair of electrodes;

b) using the pair of electrodes to detect a change in conductivity within the Debye length of the particle upon incorporation of at least one nucleotide into a growing nucleic acid strand that is complementary to the nucleic acid template;

c) washing said nanosensor array to remove excess nucleotides; and

d) repeating steps a, b, and c to obtain sequence information for said nucleic acid template.

2. The method of claim 1, wherein the nucleotides are each modified with a charge modifying structure that is associated, bound, or conjugated to the base of the nucleotide.

3. The method of claim 1, wherein the nucleotides are non-natural nucleotides.

4. The method of claim 1, wherein the nucleotides are reversibly terminated nucleotides.

5. The method of claim 1, wherein the fluid further comprises a polymerase.

6. The method of claim 5, wherein the at least one nucleotide is incorporated with the aid of said polymerase.

7. The method of claim 5, wherein the electrodes are in contact with a solution having the particle.

8. The method of claim 1, wherein said electrodes are electrically separated in the absence of said particle proximate to said nanosensor.

9. The method of claim 1, wherein b) comprises detecting a change in impedance resulting from said change in conductivity by determining current during a voltage sweep.

10. A method for sequencing a nucleic acid template, comprising:

a) contacting the nucleic acid template hybridized to a primer and coupled to a particle, which particle is proximate to a nanosensor in a nanosensor array, with a fluid containing nucleotides, wherein said nanosensor comprises a pair of electrodes;

b) using the pair of electrodes to detect a local change in charge within the Debye length of the particle upon incorporation of at least one nucleotide into a growing nucleic acid strand that is complementary to the nucleic acid template;

c) washing said nanosensor array to remove excess nucleotides; and

d) repeating steps a, b, and c to obtain sequence information for said nucleic acid template.

11. The method of claim 10, wherein the nucleotides are each modified with a charge modifying structure that is associated, bound, or conjugated to the base of the nucleotide.

12. The method of claim 10, wherein the nucleotides are non-natural nucleotides.

13. The method of claim 10, wherein the nucleotides are reversibly terminated nucleotides.

14. The method of claim 10, wherein the at least one nucleotide is incorporated with the aid of a polymerase.

15. The method of claim 10, wherein the electrodes are in contact with a solution having the particle.

16. The method of claim 15, wherein said electrodes are electrically separated in the absence of said particle proximate to said nanosensor.

17. The method of claim 10, wherein b) comprises detecting a change in impedance resulting from said local change in charge.