Rapid polymer sequencer

• US 7,949,472 B1
• Filed: 06/24/2004
• Issued: 05/24/2011
• Est. Priority Date: 06/24/2004
• Status: Expired due to Fees

• Current Assignee: USA As Represented By The Administrator Of The NASA
• Original Assignee: The United States Of America As Represented By The National Aeronautics And Space Administration
• Inventors: Brock, Mathew W., Stolc, Viktor
• Primary Examiner(s): Dejong; Eric S
• Application Number:
  US10/885,537
• Time in Patent Office: 2,525 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 or transverse direction at the tip, a polymer sequence is passed through the tip, and a change in an electrical current signal is measured as each polymer component passes through the tip. Each measured change in electrical current signals is compared with a database of reference signals, with each reference signal identified with a polymer component, to identify the unknown polymer component. The tip 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 for obtaining a reading of a sequence of components in a polymer molecule, the method comprising:

providing a pipette having a longitudinal axis and having a tapered region and having a pore with a selected pore diameter in a range of 1-40 nanometers (nm);

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

impressing a selected voltage difference across the selected liquid within the pipette pore substantially parallel to the pipette longitudinal axis direction, and providing an ionic current value induced in the selected liquid;

passing a polymer molecule, having a sequence of two or more polymer components, through the pore, and determining a change in the ionic current signal induced by passage of each of the polymer components through the pore;

comparing each of the sequence of changes in ionic current signals with each of N reference ionic current change signals (N≧

2), with each reference ionic current change signal corresponding to a reference polymer component drawn from a reference group of polymer components; and

for at least one of the determined changes in ionic current signals for one of the polymer components, identifying the polymer component with the reference polymer component whose reference ionic current change signal is closest to the at least one determined ionic current change signal.

2. 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.

3. The method of claim 2, 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.

4. The method of claim 1, 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.

5. The method of claim 1, further comprising selecting material for said pipette from a group of materials including quartz glass, aluminosilicate glass and borosilicate glass.

6. The method of claim 1, 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.

7. The method of claim 1, further comprising providing a selected second liquid in contact with a portion of a surface of said pipette not including said interior surface of said pore.

8. The method of claim 7, further comprising choosing said second liquid to be substantially the same as said selected first liquid.

9. The method of claim 7, further comprising choosing said second liquid to be different from said selected first liquid.

10. The method of claim 1, wherein said process of identifying said polymer component with said reference polymer component whose reference ionic current signal is closest to said at least one determined ionic current signal comprises:

(i) measuring a value CIC(t_m;

meas) of said determined ionic current change signal at each of a sequence of times t=t_m (m=1, . . . , M;

M≧

1);

(ii) forming an error value, defined as

11. The method of claim 1, further comprising passing said polymer sequence through said tapered tip of said pipette at an average rate in a range of 10-1000 polymer components per millisecond.

12. The method of claim 1, 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 substantially monotonically with time;

(iii) decreases substantially monotonically with time;

(iv) is substantially a step function in time;

(v) varies substantially sinusoidally with time; and

(vi) varies substantially trapezoidally with time.

13. A method for obtaining a reading of a sequence of components in a polymer molecule, the method comprising:

providing a pipette having a longitudinal axis and having a tapered region and having a pore with a selected pore diameter in a range of 1-40 nanometers (nm), where a self-assembling monolayer of a selected substance is provided on an interior surface of the pore;

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

impressing a selected voltage difference across the selected liquid within the pipette pore transverse to the pipette longitudinal axis direction, and providing an electronic current value induced in the selected liquid;

passing a polymer molecule, having a sequence of two or more polymer components, through the pore, and determining a change in the electronic current signal induced by passage of each of the polymer components through the pore

comparing each of the sequence of changes in electronic current signals with each of N reference electronic current change signals (N≧

2), with each reference electronic current change signal corresponding to a reference polymer component drawn from a reference group of polymer components; and

for at least one of the determined changes in electronic current signals for one of the polymer components, identifying the polymer component with the reference polymer component whose reference electronic current change signal is closest to the at least one determined electronic current change signal.

14. The method of claim 13, wherein where a self-assembling monolayer of a selected substance is provided on a selected portion of said interior surface of said pore.

15. The method of claim 14, 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.

16. The method of claim 13, 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.

17. The method of claim 13, further comprising selecting material for said pipette from a group of materials including quartz glass, aluminosilicate glass and borosilicate glass.

18. The method of claim 13, further comprising choosing said selected liquid to include at least one of an alkali halide, an alkaline halide, a nitrate and a sulfate.

19. The method of claim 13, further comprising providing a selected second liquid in contact with a portion of a surface of said pipette not including said interior surface of said pore.

20. The method of claim 19, further comprising choosing said second liquid to be substantially the same as said selected first liquid.

21. The method of claim 19, further comprising choosing said second liquid to be different from said selected first liquid.

22. The method of claim 13, wherein said process of identifying said polymer component with said reference polymer component whose reference electronic current change signal is closest to said at least one determined electronic current signal comprises:

(i) measuring a value CEC(t_m;

meas) of said determined electronic current change signal at each of a sequence of times t=t_m, (m=1, . . . , M;

M≧

1);

(ii) forming an error value, defined as

23. The method of claim 13, further comprising passing said polymer sequence through said tapered tip of said pipette at an average rate in a range of 10-1000 polymer components per millisecond.

24. The method of claim 13, 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 substantially monotonically with time;

(iii) decreases substantially monotonically with time;

(iv) is substantially a step function in time;

(v) varies substantially sinusoidally with time; and

(vi) varies substantially trapezoidally with time.