Electrochemical denaturation of double-stranded nucleic acid

Details Electrochemical denaturation of double-stranded nucleic acid Electrochemical denaturation of double-stranded nucleic acid

2000-03-06

2001-09-18

Campbell, Eggerton A. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

Reexamination Certificate

active

06291185

ABSTRACT:

This invention relates to processes for the treatment of nucleic acid material in order to effect a complete or partial change from double-stranded form to single-stranded form and to processes of amplifying or detecting nucleic acids involving such denaturation processes.
Double-stranded DNA (deoxyribonucleic acid) and DNA/RNA (ribonucleic acid) and RNA/RNA complexes in the familiar double helical configuration are stable molecules that, in vitro, require aggressive conditions to separate the complementary strands of the nucleic acid. Known methods that are commonly employed for strand separation require the use of high temperatures of at least 60° C. and often 100° C. for extended periods of ten minutes or more or use an alkaline pH of 11 or higher. Other methods include the use of helicase enzymes such as Rep protein of
E. coli
that can catalyse the unwinding of the DNA in an unknown way, or binding proteins such as 32-protein of
E. coli
phage T4 that act to stabilise the single-stranded form of DNA. The denatured single stranded DNA produced by the known processes of heat or alkali treatment is used commonly for hybridisation studies or is subjected to amplification cycles.
Such separation is a prerequisite of a number of protocols involving the in vitro manipulation of nucleic acids, one example of which is a reaction that produces multiple copies of target sequences of DNA and which employs a heat-stable polymerase enzyme (U.S. Pat. No. 4,683,202, K. B. Mullis et al) . This development, known as the polymerase chain reaction (PCR), is of significant commercial importance and strand separation is normally effected by heating the sample to approximately 95° C. The removal of the need to heat the sample would provide a number of benefits. For example, it allows the design of compact and readily controllable apparatus, and the use of higher fidelity mesophilic enzymes.
WO 92/04470 discloses a process whereby nucleic acid strands are separated by the application of an electric field. The advantages of the electrical method are discussed in greater detail, along with the method's application in amplification reactions such as PCR and ligase chain reaction. Forms of electrochemical cells for carrying out the reaction are described and also the use of “promoter” compounds that enhance the efficiency of denaturation.
Prior to WO92/04470, a number of other workers had described denaturation of DNA in electrochemical cells. However, in none of these cases was single-stranded product left free in solution in useful quantities. Rather, DNA appears to have become irreversibly bound to the surface of the electrode, in which condition it is not available for further participation in processes such as PCR. In the method of electrical denaturation described in WO92/04470, single strands accumulate in solution and their utility and integrity is confirmed by subsequently performing PCR.
In WO92/04470 electrical denaturation of DNA was carried out using an electrode comprising a central rod of glassy carbon encased in a teflon sleeve except at its end. The working electrode was of platinum mesh lying against the teflon sleeve. A calomel reference electrode was used, situated in a side chamber which was connected to the main cell by a capillary tube (see Stanley C. J. et al, J. Immunol. Meth. [1988], 112, 153-161). Using this apparatus the most rapid denaturation was achieved in 15 minutes with the working electrode at a potential of −1V with respect to the reference. The presence of NaCl in the reaction delayed denaturation.
In WO92/04470, a PCR reaction is conducted in which there are repeated denaturation operations conducted using the electro-chemical cell described with intervening amplification stages. The denaturation stages are each conducted for a period of five minutes or longer and the total time for the PCR reaction is therefore very extended. Furthermore, the conditions under which the PCR reaction was conducted electrochemical in WO92/00470 differ from those of the conventional PCR process in that it was not found possible to use a conventional PCR buffer system. In order to obtain denaturation, it was necessary to conduct the process at a much lower ionic strength than would be consistent with such a buffer system. Excluding the promoter methyl viologen, the process was basically conducted in distilled water.
It has now been discovered that it is now possible to conduct a denaturation electrochemically considerably faster than is disclosed in WO92/04470 and to conduct an amplification procedure much faster than is disclosed there.
Although the spacing between the two working electrodes in WO92/04470 is not explicitly stated, it was in fact several millimetres.
Accordingly, the present invention provides a process for denaturing double-stranded nucleic acid which comprises subjecting a solution containing said nucleic acid to a voltage applied between electrodes under conditions such as to convert at least a portion of said nucleic acid to a wholly or partially single-stranded form in the solution, wherein said electrodes approach to within 1.5 mm of one another in said solution. Preferably, the electrodes approach more closely, e.g. within 1 mm or more preferably 0.5 mm of one another. Ideally, the electrodes are adjusted to be spaced by as little as possible whilst ensuring that they do not contact one another to produce a short circuit.
It is preferred to apply a voltage difference of from 0.5 to 3 volts between the electrodes. Voltage differences above 3 volts seem to inhibit denaturation although the mechanism involved here is presently unknown.
Preferably, the process is conducted at a voltage of 1.5 to 2.5 volts measured as a voltage difference between the electrodes.
Preferably, where the electrodes most closely approach one another, one or both of the electrodes is pointed. Such an electrode may be provided with a single point or a plurality of points. There appears to be some inter-relationship between the ideal voltage applied and the shape of the electrode and it may be that there is a preferred or ideal field gradient at the point of the electrode which can be achieved by adjustment of the voltage to suit the sharpness of the part of the electrode at which the denaturation takes place. Optionally, one can conduct the denaturation using a constant current supply rather than a regulated voltage and this may serve to compensate for variations in the geometrical set-up of the electrodes between different denaturation operations.
Where a constant current regime is employed, it will generally be preferable to use a current of from 80 to 160 &mgr;A, e.g. about 100 to 125 &mgr;A.
As described in WO92/04470, one may employ a promoter compound such as methyl viologen to produce more rapid denaturation. Other promoters are described in WO93/15224, i.e. multivalent cations such as magnesium. Other multi-valent cations which are effective and which can be used include lanthanum (La
3+
). The cations used as the promoters may include inorganic cations complexed with inorganic or organic ligands, e.g. Pt(NH
3
)
6
4+
and Cr(NH
3
)
6
2+
.
The promoter may be any inorganic or organic molecule which increases the rate of extent of denaturation of the double helix. It should be soluble in the chosen reaction medium. It preferably does not affect or interfere with DNA or other materials such as enzymes or oligonucleotide probes which may be present in the solution. Alternatively, the promoter may be immobilised to or included in material from which the electrode is constructed.
The promoter may be a water-soluble compound of the bipyridyl series, especially a viologen such as methyl-viologen or a salt thereof. Whilst the mechanism of operation of such promoters is presently not known with certainty, it is believed that the positively charged viologen molecules interact between the negatively charged nucleic acid such as DNA and the negatively charged cathode to reduce electrostatic repulsion therebetween and hence to promote the approach of the

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