Disassociation of interacting molecules

Details Disassociation of interacting molecules Disassociation of interacting molecules

2000-03-31

2001-12-25

Whisenant, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S287200, C435S091200, C436S094000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

active

06333157

ABSTRACT:

This invention relates to processes for the treatment of interacting molecules in order to effect a complete or partial disassociation thereof.
Double-stranded DNA (deoxyribonucleic acid) and DNA/RNA (ribonucleic acid) and RNA/RNA complexes in the familiar double helical configuration are produced by the stable interaction of single-stranded molecules. Such complexes 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. or use an alkaline pH of 11 or higher or a low pH. 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 electrochemical 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 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.
In WO95/25177 we showed it is possible to conduct a denaturation electrochemically considerably faster than is disclosed in W092/04470 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 millimeters.
An improved method is described in WO95/25177 in which a solution containing said nucleic acid is subjected to a voltage applied between electrodes which approach to within 1.5 mm of one another in said solution. This results in a substantial increase in the rate of denaturation such that WO95/25177 contains examples in which complete denaturation of DNA is achieved within 1 to 2 minutes in comparison with denaturation times of at least 25 minutes using the electrode set up of WO92/04470.
It is indicated in WO95/25177 that rather than simply turning the electrical field on and off when conducting PCR using the apparatus described there, one may optionally reverse the field. In WO95/25177, this reversal of the field is seen as being merely an equivalent to turning the field off. In Application GB96139803, periods of zero voltage are used in combination with such field reversals, to further improve the process.
Although the process of Application WO92/04470 can take place in a solution containing only the electrode and the nucleic acid dissolved in water containing a suitable buffer, the process can be facilitated by the presence in the solution containing the nucleic acid of a promoter compound. Methyl viologen or a salt thereof was disclosed as the preferred promoter compound.
It is believed that the positively charged viologen molecules interact between the negatively charged DNA and the negatively charged cathode to reduce electrostatic repulsion therebetween and hence to promote the approach of the DNA to the electrode surface where the electrical field is at its strongest. Accordingly, we expressed a preference in WO92/04470 to employ as promoters compounds having spaced positively charged centres, e.g. bipolar positively charged compounds. Preferably, the spacing between the positively charged centres was to be similar to that in viologens.
WO93/15224 was in turn based on the discovery that multivalent inorganic cations, preferably Mg
2+
, can also act as promoters in such a system with approximately the same efficacy as methyl viologen.
It is thought that large cations such as Mg
2+
are able to act as a bridge between a negative electrode and negatively charged regions of the double-stranded nucleic acid.
As described in GB9614544.6, it has also been found that lithium ions can also promote denaturation.
The concentration of said promoter cation is preferably from 1 Mm to 50 Mm, more preferably from 5 Mm to 20 Mm, e.g. about 10 Mm.
The rate and extent of denaturation obtainable in such electrochemical systems depends on a number of factors, including the medium in which the nucleic acid is present. Processes used in molecular biology such as nucleic acid hybridisation assays or amplification procedures like PCR are conducted in media containing a buffering agent to maintain optimum Ph for the reactions involved. However, the presence of such a buffering agent is generally adverse to the electrochemical dehybridisation of nucleic acids. This is to some extent overcome by an appropriate choice of promoter, as described above, but it would be highly desirable to develop systems in which the presence of the buffer was substantially less adverse in its impact on the dehybridisation process.
Thus, whilst it is normally found that increasing ionic strength tends to stabilise the interaction between molecules, so that disassociation occurs more readily in the absence of buffers, which are a source of ions contributing to

Affiliated with

Also associated with

Rating

Disassociation of interacting molecules does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Disassociation of interacting molecules, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Disassociation of interacting molecules will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2571573