Disassociation of interacting molecules

• US 6,333,157 B1
• Filed: 03/31/2000
• Issued: 12/25/2001
• Est. Priority Date: 04/02/1997
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Bergmann, Karin, Watson, Susan L., Miller-Jones, David N.
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/381,896
• Time in Patent Office: 634 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.19
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

dsDNA or other interacting molecules, e.g. antibody and antigen, are disassociated by applying an electrical voltage to a solution containing said molecules in a buffer favoring said disassociation e.g. CHES, CAPS OR CAPSO.

Claims

1. A process for denaturing nucleic acid molecules, comprising subjecting a liquid containing said nucleic acid molecules to an electrical voltage applied between electrodes under conditions such as to wholly or partially denature at least a proportion of said nucleic acid molecules in the presence of a buffer selected from the group consisting of N-cyclohexyl-2-aminoethane sulphonic acid (CHES), N-cyclohexyl-3-aminoethane sulphonic acid (CAPS), and N-cyclohexyl-3-amino-2-hydroxypropane sulphonic acid (CAPSO).

2. The process of claim 1, wherein said buffer has a pK_a of not less than 8.5.

3. The process of claim 1, wherein said buffer has a pK_a of not less than 9.0.

4. The process of claim 1, wherein said buffer has a pK_a of not less than 9.4.

5. The process of claim 1, conducted at a pH of from 7 to 9.

6. The process of claim 1, conducted in from a 5 to 10 mM concentration of said buffer.

7. The process of claim 6, wherein the electrodes approach to within 0.5 mm to one another.

8. The process of claim 1, wherein a voltage of from 0.5 to 3 volts is applied between said electrodes.

9. The process of claim 8, wherein a voltage of from 1.5 to 2.5 volts is applied between said electrodes.

10. A process of repeated denaturation of nucleic acid molecules, wherein said nucleic acid molecules are denatured by a process comprising subjecting a liquid containing said nucleic acid molecules to an electrical voltage applied between electrodes under conditions such as to wholly or partially denature at least a proportion of said nucleic acid molecules in the presence of a buffer selected from the group consisting of N-cyclohexyl-2-aminoethane sulphonic acid (CHES), N-cyclohexyl-3-aminoethane sulphonic acid (CAPS), and N-cyclohexyl-3-amino-2-hydroxypropane sulphonic acid (CAPSO), in which said voltage is applied as a repeating pulse having a duration of up to 2 minutes.

11. The process of claim 10, wherein said voltage is applied as repeating pulse having a duration of up to 1 minute.

12. The process of claim 10, wherein between said pulses the voltage is turned off and/or reversed for a period equal to the period for which the voltage is applied.

13. The process of claim 12, wherein said voltage is applied as pulses at a frequency of from 0.01 to 10 Hz.

14. The process of claim 10, wherein said voltage is applied such that there are, in any order, periods of application of voltage with a first polarity, periods of application of voltage with the opposite polarity to said first polarity and periods of reduced applied voltage.

15. The process of claim 14, wherein said cycles are from 1 second to 5 minutes in length.

16. The process of claim 15, wherein the periods during which said voltage is applied with a first polarity and said periods during which said voltage is applied with a second polarity are each independently of from 0.5 seconds to 1 minute.

17. The process of claim 15, wherein the periods during which said voltage is reduced are each individually from 0.5 seconds to 3 minutes.

18. A process of amplifying a target sequence of nucleic acid comprising hybridisation, extension and denaturation of nucleic acid wherein said denaturation is conducted by subjecting said nucleic acid to a voltage applied between electrodes in the presence of a buffer selected from the group consisting of N-cyclohexyl-2-aminoethane sulphonic acid (CHES), N-cyclohexyl-3-aminoethane sulphonic acid (CAPS), and N-cyclohexyl-3-amino-2-hydroxypropane sulphonic acid (CAPSO).

19. The process of claim 18, which is a PCR or LCR amplification.

20. The process of claim 18, wherein said buffer has a pK_a of not less than 8.5.

21. The process of claim 18, wherein said buffer has a pK_a of not less than 9.0.

22. The process of claim 18, wherein said buffer has a pK_a of not less than 9.4.

23. The process of claim 18, conducted at a pH of from 7 to 9.

24. The process of claim 18, conducted in from a 5 to 10 mM concentration of said buffer.

25. The process of claim 24, wherein the electrodes approach to within 0.5 mm to one another.

26. The process of claim 18, wherein a voltage of from 0.5 to 3 volts is applied between said electrodes.

27. The process of claim 26, wherein a voltage of from 1.5 to 2.5 volts is applied between said electrodes.

28. A kit for use in a process of denaturing nucleic acid molecules which comprises an electrode, a counter electrode, optionally a reference electrode, and a buffer selected from the group consisting of N-cyclohexyl-2-aminoethane sulphonic acid (CHES), N-cyclohexyl-3-aminoethane sulphonic acid (CAPS), and N-cyclohexyl-3-amino-2-hydroxypropane sulphonic acid (CAPSO).