Rapid polymer sequencer

• US 8,494,782 B1
• Filed: 04/21/2011
• Issued: 07/23/2013
• Est. Priority Date: 06/24/2004
• Status: Active Grant

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: The United States of America As Represented By The Secretary of Agriculture
• Inventors: Stolc, Viktor, Brock, Matthew W
• Primary Examiner(s): Dejong, Eric S
• Application Number:
  US13/092,048
• Time in Patent Office: 824 Days
• Field of Search: None
• US Class Current: 702/19
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Method and system for rapid and accurate determination of each of a sequence of unknown polymer components, such as nucleic acid components. A self-assembling monolayer of a selected substance is optionally provided on an interior surface of a pipette tip, and the interior surface is immersed in a selected liquid. A selected electrical field is impressed in a longitudinal direction, or in a transverse direction, in the tip region, a polymer sequence is passed through the tip region, and a change in an electrical current signal is measured as each polymer component passes through the tip region. Each of the measured changes in electrical current signals is compared with a database of reference electrical change signals, with each reference signal corresponding to an identified polymer component, to identify the unknown polymer component with a reference polymer component. The nanopore preferably has a pore inner diameter of no more than about 40 nm and is prepared by heating and pulling a very small section of a glass tubing.

Claims

1. A method of fabricating a nanopore, the method comprising:

heating a hollow cylinder of a pipette material, comprising primarily at least one of quartz glass, aluminosilicate glass and borosilicate glass, by a process comprising use of at least one of a laser, an infrared light source and a heated metal for heating one or more locations on the cylinder for a first time interval, the hollow cylinder having first and second cylinder ends, having a longitudinal axis and having a non-zero initial inner diameter; and

applying a machine controlled translation force to translate at least one of the first and second cylinder ends relative to the other of the first and second cylinder ends by a change in end-to-end separation distance no greater than about 2 cm during a second time interval that partly or wholly overlaps the first time interval, in order to encourage the hollow cylinder to separate into at least first and second pipettes, each with a corresponding nanopore, with at least one pore diameter in a range of 1-40 nanometers (nm) and with at least one pore length no greater than about 2 cm.

3. The method of claim 1, further comprising:

providing a selected liquid in contact with an interior surface of said pore;

impressing a non-zero voltage difference across the selected liquid within said pore approximately parallel to a longitudinal axis direction of said cylinder, and determining at least one of an electrical current value and an ionic current value induced in the selected liquid; and

passing a polymer molecule, having a sequence of polymer components, through said pore in a first direction, determined with reference to the longitudinal axis direction, and determining at least one of an electrical current signal and an ionic current signal induced by passage of each of the polymer components through said pore.

4. The method of claim 3, further comprising selecting material for said pipette from a group of materials including quartz glass, aluminosilicate glass and borosilicate glass.

5. The method of claim 3, further comprising passing said polymer sequence through said pore at an average rate in a range of 1-1000 polymer components per msec.

6. The method of claim 3, further comprising choosing said polymer sequence to be a nucleic acid sequence including the bases adenine, cytosine and guanine and at least one of the bases thymine and uracil.

7. The method of claim 3, further comprising selecting said voltage difference from a group of time-dependent differences including a difference that (i) is approximately uniform in time;

(ii) increases monotonically with time;

(iii) decreases monotonically with time;

(iv) is a step function in time;

(v) varies sinusoidally with time; and

(vi) varies trapezoidally with time.

8. The method of claim 3, further comprising choosing said selected liquid to include at least one of an alkali halide, an ammonium compound, an ionic organic compound and an ionic inorganic compound.

9. The method of claim 1, further comprising providing a self-assembling monolayer of a selected substance on a selected portion of said interior surface of said pore.

10. The method of claim 9, further comprising choosing said self assembling monolayer to include at least one of:

(i) octadecyltrichlorosilane on glass and (ii) (16-Mercapto)hexadecanoic acid on a gold substrate.

11. The method of claim 1, further comprising:

providing a selected liquid in contact with an interior surface of said pore;

impressing a non-zero voltage difference across the selected liquid within said pore transverse to a longitudinal axis direction of said cylinder, and determining an ionic current value induced in the selected liquid; and

passing a polymer molecule, having a sequence of polymer components, through said pore in a first direction, determined with reference to the longitudinal axis direction, and determining an ionic current signal induced by passage of each of the polymer components through said pore.

12. The method of claim 11, further comprising passing said polymer sequence through said pore at an average rate in a range of 1-1000 polymer components per msec.

13. The method of claim 11, further comprising choosing said polymer sequence to be a nucleic acid sequence including the bases adenine, cytosine and guanine and at least one of the bases thymine and uracil.

14. The method of claim 11, further comprising selecting said voltage difference from a group of time-dependent differences including a difference that (i) is substantially uniform in time;

(ii) increases monotonically with time;

(iii) decreases monotonically with time;

(iv) is a step function in time;

(v) varies sinusoidally with time; and

(vi) varies trapezoidally with time.

15. The method of claim 11, further comprising choosing said selected liquid to include at least one of an alkali halide, an ammonium compound, an ionic organic compound and an ionic inorganic compound.

16. The method of claim 1, further comprising providing a self-assembling monolayer of a selected substance on a portion of said interior surface of said pore.

17. The method of claim 16, further comprising choosing said self assembling monolayer to include at least one of:

(i) octadecyltrichlorosilane on glass and (ii) (16-Mercapto)hexadecanoic acid on a gold substrate.

18. The method of claim 1, further comprising applying said machine controlled translation force in a range of between 10 dynes and 10 million dynes in a direction corresponding to said longitudinal axis.

2. The method of 1, further comprising choosing said heating source from a group of heating sources consisting of a laser, an infrared light source, and a heated metal.