IP Library Granted Patent US 9,410,171
Granted Patent B2
US 9,410,171 · App. 13/923,337 · Granted Aug 9, 2016

Non-thermal cycling for polymerase chain reaction

Inventors: Inanc Ortac (La Jolla, CA); Sadik C. Esener (Solana Beach, CA)
Assignee: The Regents of the University of California
C12P19/34C12Q1/686
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Quick Facts
Patent No.
US 9,410,171
App. No.
13/923,337
Granted
Aug 9, 2016
Kind
B2
Abstract

Techniques, systems, and devices are disclosed for non-thermal cycling of polymerase chain reaction (PCR). In one aspect, a method for cycling PCR includes receiving an electrolytic fluid including ions, primers, polymerase enzymes, nucleotides, and a double-stranded nucleic acid in a fluid chamber having a first electrode and a second electrode, applying an electric field across the first and the second electrodes to generate a first pH level of the electrolytic fluid to denature the double-stranded nucleic acid to at least partial single strands, and applying a second electric field across the first and second electrodes to produce a second pH level of the electrolytic fluid, in which the second pH level enables binding of a polymerase enzyme and a primer with a corresponding segment of the single strands.

Claims (32)

1. A method for polymerase chain reaction (PCR) cycling, comprising:

receiving an electrolytic fluid including ions, primers, polymerase enzymes, nucleotides, and a double-stranded nucleic acid in a fluid chamber having a first electrode and a second electrode located on two opposing sides of the electrolytic fluid;

applying an electric field across the first and the second electrodes to generate a first pH level of the electrolytic fluid to denature the double-stranded nucleic acid to at least partial single strands; and

applying a second electric field across the first and second electrodes to produce a second pH level of the electrolytic fluid,

wherein the second pH level enables binding of a polymerase enzyme and a primer with a corresponding segment of the single strands.

2. The method as in claim 1 , wherein the second pH level enables synthesis of new complementary nucleic acid strands from the nucleotides using the at least partial single strands.

3. The method as in claim 1 , further comprising applying a third electric field across the first and second electrodes to produce a third pH level of the electrolytic fluid, wherein the third pH level enables synthesis of new complementary nucleic acid strands using the at least partial single strands.

4. The method as in claim 1 , wherein the fluid chamber includes a semi-permeable membrane separating a first region including the first electrode from a second region including the second electrode, the semi-permeable membrane permeable for the ions to traverse the membrane and impermeable to the primers, polymerase enzymes, nucleotides, and double-stranded nucleic acid.

5. The method as in claim 4 , wherein the receiving the electrolytic fluid includes adding the primers, polymerase enzymes, nucleotides, and double-stranded nucleic acid into the first region or into the second region of the fluid chamber.

6. The method as in claim 5 , further comprising moving the semi-permeable membrane to reduce the volume of the region that contains the primers, polymerase enzymes, nucleotides, and double-stranded nucleic acid.

7. The method as in claim 1 , wherein the polymerase enzymes are covalently attached to a surface of the fluid chamber.

8. The method as in claim 1 , wherein the applied electric fields create a spatial ion distribution in the electrolytic fluid based on at least one of a magnitude of the electric field, ion concentration, or dimension and geometry of the electrodes, the spatial ion distribution including a first layer of first ions electrically attracted to an oppositely-charged electrode and a second layer of second ions electrically attracted to the first layer.

9. The method as in claim 1 , wherein ions include H + , OH − and Mg 2+ .

10. The method as in claim 1 , further comprising selecting a magnitude and duration of the applied electric fields.

11. A method of polymerase chain reaction cycling, the method comprising:

a) receiving an electrolytic fluid in a fluid chamber having a first electrode and a second electrode in predetermined geometric configuration, the electrolytic fluid comprising ions, at least one primer, at least on polymerase enzyme, an excess amount of nucleotides, and a double-stranded nucleic acid;

b) applying a first electric field between the first electrode and the second electrode to produce a first pH level of the electrolytic fluid;

c) applying a second electric field between the first electrode and second electrode to produce a second pH level of the electrolytic fluid; and

d) maintaining the second pH level of the electrolytic fluid for a predetermined period of time,

wherein the first pH level enables denaturing of the double-stranded nucleic acid to at least partial single strands,

wherein the second pH level enables binding of a polymerase enzyme and a primer with a corresponding segment of the single strands, and

wherein the maintaining of the second pH level enables synthesis of new complementary nucleic acid strands based on the single strands.

12. The method of claim 11 , wherein actions (b) through (d) are repeated at least once wherein the double-stranded nucleic acid is amplified.

13. A method of polymerase chain reaction cycling, the method comprising:

a) receiving an electrolytic fluid in a fluid chamber having a first electrode and a second electrode in predetermined geometric configuration, the electrolytic fluid comprising ions, at least one primer, at least one polymerase enzyme, an excess amount of nucleotides, and a double-stranded nucleic acid;

b) applying a first electric field between the first electrode and the second electrode to produce a first pH level of the electrolytic fluid;

c) applying a second electric field between the first electrode and second electrode to produce a second pH level of the electrolytic fluid; and

d) applying a third electric field between the first electrode and the second electrode to produce a third pH level of the electrolytic fluid,

wherein the first pH level enables denaturing of the double-stranded nucleic acid to at least partial single strands,

wherein the second pH level enables binding of a polymerase enzyme and a primer with a corresponding segment of the single strands, and

wherein the third pH level enables synthesis of new complementary nucleic acid strands based on the single strands.

14. The method of claim 13 , wherein actions (b) through (d) are repeated at least once wherein the double-stranded nucleic acid is amplified.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 19, 2015
From: ORTAC, INANC; ESENER, SADIK C
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 034982/0960 →
Continuity (2)
Provisional Application 61662301 · Jun 20, 2012
Related Publication 20130344539A1 · Dec 26, 2013