Electrochemical denaturation of double-stranded nucleic acid

• US 6,395,489 B1
• Filed: 05/10/2000
• Issued: 05/28/2002
• Est. Priority Date: 09/12/1990
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Stanley, Christopher J.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/568,622
• Time in Patent Office: 748 Days
• Field of Search: 435/6,91.1,91.2 436/94
• US Class Current: 435/6.12
• 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 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A process is described for denaturing native double-stranded nucleic acid material into its individual strands in an electrochemical cell. The process disclosed is an electrical treatment of the nucleic acid with a voltage applied to the nucleic acid material by an electrode. The process may also employ a promoter compound such as methyl viologen to speed denaturation. The process may be used in the detection of nucleic acid by hybridizing with a labelled probe or in the amplification of DNA by a polymerase chain reaction or ligase chain reaction.

Claims

1. A process for denaturing double-stranded nucleic acid, wherein one strand of said double-stranded nucleic acid is bound to an electrode surface and the other strand of said double-stranded nucleic acid is not so bound, comprising the steps of:

applying a voltage to said double-stranded nucleic acid with said electrode; and

converting at least a proportion of said nucleic acid to a wholly or partially single-stranded form so as to produce single-stranded nucleic acid in solution, which single-stranded nucleic acid is capable of renaturation to double-stranded form.

2. The process according to claim 1, wherein said electrode is immersed in a solution and a potential of from −

0.5 to −

1.5 V is applied to said electrode with respect to said solution.

3. The process according to claim 2 wherein said voltage is from −

0.8 to −

1.1 V.

4. The process according to claim 2 wherein the ionic strength of said solution is no more than 250 mM.

5. The process according to claim 2 wherein the ionic strength of said solution is n more than 100 mM.

6. The process according to claim 2 wherein the ionic strength of said solution is no more than 50 mM.

7. The process according to claim 2 wherein the ionic strength of said solution is no more than 25 mM.

8. The process according to claim 2 wherein the ionic strength of said solution is no more than 5 mM.

9. The process according to claim 1 wherein the denaturation is conducted in the presence of a promoter compound which assists said denaturation.

10. The process according to claim 9, wherein said compound is methyl viologen or a salt thereof.

11. The process according to claim 1 wherein the electrode is constructed of carbon, gold or platinum.

12. The process according to claim 1 wherein said process is carried out at a temperature above ambient temperature but less than the melting point of said double-stranded nucleic acid.

13. The process according to claim 1 wherein said process is carried out at ambient temperature.

14. The process according to claim 1 wherein said process is carried out at a pH of from 3 to 10.

15. The process according to claim 14 wherein said process is carried out at a neutral pH.

16. The process according to claim 1 wherein said denaturation is conducted with said electrode immersed in an aqueous solution containing a buffer whose nature and ionic strength are such as not to interfere with strand separation of said nucleic acid.

17. The process according to claim 1 wherein said nucleic acid is DNA.

18. The process according to claim 1 wherein said process is carried out as a denaturing step in a nucleic acid amplification procedure.

19. A process for the annealing of complementary single strands of nucleic acid into double-stranded form comprising the steps of:

applying a voltage to said complementary single strands of nucleic acid with an electrode; and

converting at least a proportion of said complementary single strands to double-stranded nucleic acid which is capable of denaturation into single stranded form.

20. The process according to claim 19, wherein one of said complementary strands of nucleic acid is immobilized to said electrode.

21. A process for reversibly hybridizing or annealing single-stranded nucleic acid in solution to nucleic acid of complimentary sequence thereto which is immobilized on a surface of an electrode, comprising applying either a negative voltage to denature nucleic acids or a positive voltage to renature nucleic acids.

22. The process according to claim 21, wherein the ionic strength of said solution is no more than 250 mM.

23. The process according to claim 22, wherein said ionic strength is no more than 100 mM.

24. The process according to claim 22, wherein said ionic strength is no more than 50 mM.

25. The process according to claim 22, wherein said ionic strength is no more than 25 mM.

26. The process according to claim 22, wherein said ionic strength is no more than 5 mM.

27. The process according to claim 21, wherein said electrode is constructed of carbon, gold or platinum.

28. The process according to claim 21, wherein said process is carried out at a temperature above ambient temperature but less than the melting point of the double-stranded nucleic acid.

29. The process according to claim 21, wherein said process is carried out at ambient temperature.

30. The process according to claim 21, wherein said process is carried out at a pH of from 3 to 10.

31. The process according to claim 30, wherein said process is carried out at a neutral pH.

32. The process according to claim 21, wherein said nucleic acids are DNA.