Electrochemical denaturation of double-stranded nucleic acid

Claim 2

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.

Claim 3

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.

Claim 4

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

Claim 5

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.

Claim 6

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

Claim 7

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

Claim 8

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

Claim 9

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

Claim 10

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

Claim 11

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.

Claim 12

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

Claim 13

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

Claim 14

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

Claim 15

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

Claim 16

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

Claim 17

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

Claim 18

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.

Claim 19

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

Claim 20

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

Claim 21

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.

Claim 22

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.

Claim 23

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

Claim 24

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.

Claim 25

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.

Claim 26

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

Claim 27

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.

Claim 28

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.

Claim 29

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.

Claim 30

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.

Claim 31

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.

Claim 32

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.

Claim 33

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.

Claim 34

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.

Claim 35

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