Electrochemical denaturation of double-stranded nucleic acid

• US 5,527,670 A
• Filed: 08/09/1994
• Issued: 06/18/1996
• Est. Priority Date: 09/12/1990
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Scientific Generics Limited (Science Group Plc)
• Inventors: Stanley, Christopher J.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/288,231
• Time in Patent Office: 679 Days
• Field of Search: 435/6, 435/91.2, 435/91.52, 436/63, 436/94, 935/78
• 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 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 promotor 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 comprising the steps of:

applying a voltage to a solution containing said nucleic acid with an 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 said solution not bound to said electrode, which single-stranded nucleic acid is capable of renaturation to double-stranded form.

2. A process as claimed in claim 1, wherein a potential of from -0.5 to -1.5 V is applied to said electrode with respect to said solution.

3. A process as claimed in claim 2, wherein said voltage is from -0.8 to -1.1 V.

4. A process as claimed in claim 1, further comprising the steps of:

contacting said solution with said electrode, a reference electrode and a counter-electrode; and

applying a voltage between said electrode and said counter-electrode so as to achieve a pre-selected voltage between said electrode and said reference electrode.

5. A process as claimed in claim 1, wherein the ionic strength of said solution is no more than 250 mM.

6. A process as claimed in claim 5, wherein the said ionic strength is no more than 100 mM.

7. A process as claimed in claim 5, wherein the said ionic strength is no more than 50 mM.

8. A process as claimed in claim 5, wherein the said ionic strength is no more than 25 mM.

9. A process as claimed in claim 5, wherein the said ionic strength is no more than 5 mM.

10. A process as claimed in claim 1, wherein said solution contains or said electrode has on its surface a promoter compound which assists said denaturization.

11. A process as claimed in claim 10, wherein said compound is methyl viologen or a salt thereof.

12. A process as claimed in claim 1, wherein said electrode is constructed of carbon, gold or platinum.

13. A process as claimed in claim 1, carried out at a temperature less than the melting point of the double-stranded nucleic acid.

14. A process as claimed in claim 13, carried out at ambient temperatures.

15. A process as claimed in claim 1 carried out at a pH of from 3 to 10.

16. A process as claimed in claim 15, carried out at a neutral pH.

17. A process as claimed in claim 1, wherein said nucleic acid is dissolved 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.

18. A process as claimed in claim 1, wherein said nucleic acid is DNA.

19. A process as claimed in claim 1, wherein said process is carried out as a denaturing step in a nucleic acid amplification procedure.

20. A process for amplifying a target sequence of nucleic acid comprising cycles of:

amplifying said target sequence of nucleic acid by hybridization, replication; and

denaturing of nucleic acid, wherein denaturation is produced by applying a voltage to a solution containing said nucleic acid with an electrode so as to produce single-stranded nucleic acid in said solution not bound to said electrode.

21. A process as claimed in claim 20, which is a polymerase chain reaction amplification process or a ligase chain reaction amplification process.

27. A process as claimed in claim 21 for amplifying a target nucleic acid comprising the steps of:

(a) providing nucleic acid of a sample as single-stranded nucleic acid;

(b) providing in the sample at least four nucleic acid probes, wherein;

i) the first and second of said probes are primary probes, and the third and fourth of said probes are secondary nucleic acid probes;

ii) the first probe is a single strand capable of hybridising to a first segment of a primary strand of the target nucleic acid;

iii) the second probe is a single strand capable of hybridising to a second segment of said primary strand of the target nucleic acid;

iv) the 5'"'"' end of the first segment of said primary strand of the target is positioned relative to the 3'"'"' end of the second segment of said primary strand of the target to enable joining of the 3'"'"' end of the first probe to the 5'"'"' end of the second probe, when said probes are hybridised to said primary strand of said target nucleic acid;

v) the third probe is capable of hybridising to the first probe; and

iv) the fourth probe is capable of hybridising to the second probe; and

(c) repeatedly or continuously;

i) hybridising said probes with nucleic acid in said sample;

ii) ligating hybridised probes to form reorganised fused probe sequences; and

iii) denaturing DNA in said sample by applying a voltage from an electrode to the reaction mixture.

22. A process for replicating a nucleic acid which comprises:

separating strands of a sample double-stranded nucleic acid in solution under the influence of an electrical voltage applied to said solution from an electrode so as to produce single-stranded denatured nucleic acid in said solution not bound to said electrode;

hybridizing said separated strands of said nucleic acid with at least one oligonucleotide primer that hybridizes with at least one of said strands of said denatured nucleic acid;

synthesizing at least one extension product of said at least one oligonucleotide primer which is sufficiently complementary to a respective strand of said nucleic acid to hybridize therewith; and

separating said at least one extension product from said nucleic acid strand with which it is hybridized to obtain said at least one extension product.

23. A process as claimed in claim 22, which further involves repeating the procedure defined in claim 22 cyclicly.

24. A process as claimed in claim 22, wherein said hybridization step is carried out using two primers which are complementary to different strands of said nucleic acid.

25. A process as claimed in claim 22, wherein the separating to obtain said at least one extension product is carried out by applying to a solution of said at least one extension product a voltage from said electrode.

26. A process as claimed in claim 22, for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate complementary strands, of equal or unequal length, which process comprises:

(a) treating the strands with two oligonucleotide primers, for each different specific sequence being applied, under conditions such that for each different sequence being amplified an extension product of each primer is synthesised which is complementary to each nucleic acid strand, wherein said primers are selected so as to be substantially complementary to different strands of each specific sequence such that the extension product synthesised from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer;

(b) separating the primer extension products from the templates on which they were synthesised to produce single-stranded molecules by applying a voltage from an electrode to the reaction mixture; and

(c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a primer extension product is synthesised using each of the single strands produced in step (b) as a template.

28. A process of detecting the presence or absence of a predetermined nucleic acid sequence in a sample which comprises:

denaturing a sample double-stranded nucleic acid by means of a voltage applied to the sample in a solution by means of an electrode so as to produce single-stranded denatured nucleic acid in said solution not bound to said electrode;

hybridizing said denatured nucleic acid with an oligonucleotide probe for said sequence; and

determining whether the said hybridization has occurred.

29. A kit for use in a process of nucleic acid amplification, said kit comprising:

an electrode,

a counter electrode, and

more than one primer for use in a PCR procedure or an LCR procedure.

30. The kit according to claim 29 further comprising a polymerase or a ligase and/or nucleotides suitable for use in a PCR process.