Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-11-01
2003-05-20
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C435S091210, C536S023100, C536S024330
Reexamination Certificate
active
06566103
ABSTRACT:
The present invention relates to a process for amplifying nucleic acid sequences by means of the polymerase chain reaction. More specifically, it relates to a process wherein the consecutive cycles of denaturation and renaturation are achieved by a controlled oscillation of the local concentration of divalent metal ions. This allows the reaction to proceed at constant temperature, and depending upon the metal ions used at lower temperatures near physiological values.
In the biotechnology and biomedical industry large number of copies of a particular gene or polynucleic acid may be needed for various purposes such as sequencing and diagnostic applications. Simple and reliable methods to generate such amounts are consequently indispensable for the success of future industrial and scientific developments. Any new technique to amplify genetic material and in particular for diagnostic applications, should minimize human intervention and chemical addition steps. A further prerequisite is that it should be easily amenable to automation.
Currently relatively large amounts of particular gene sequence can be produced by the polymerase chain reaction (EP-B-0200 362, EP-B-0201 184). A method which offered significant benefits over classical procedures such as cloning. In essence PCR is based on the repetitive thermal denaturation of double stranded (dsDNA), a process which is known as thermal cycling. As the reaction temperature is cycled between about 70° C. and 94° C., the polymerases would denature soon. In Kleppe et al., J.Mol.Biol. 56: 341 (1971), a process is described for synthesizing DNA using primer-initiated, template-directed repair replication, and it is suggested that cycles of replication could be repeated, adding every time a fresh dose of DNA polymerase, which would be an expensive procedure. A nice solution was offered by the introduction of thermostable DNA polymerases which were derived From thermophilic bacteria (e.g.
Thermus aquaticus
). Such enzymes, however, have a higher error rate than other polymerases, particularly of eukaryotic origin, which operate at lower temperatures.
The need for extreme conditions to allow separation of both strands that make us the DNA helix directly results from the fact that the DNA double helix is a relatively stable structure due to the propensity of the bases to form hydrogen bonds with each other in a very specific way. Apart from base pairing several additional conditions need to be satisfied to guarantee stability at a particular temperature. Ionic strength of the medium has a very important role and at low electrolyte concentration dsDNA is denatured due to the lack of counterions. These counterions may be mono- or divalent metal ions which stabilize the structure by binding to the phosphate moieties and effectively cancel the net negative charges preventing unwinding of the helix due to repulsive forces. However at elevated concentrations some divalent metal ions (in particular: Cu
2+
, Cd
2+
, Zn
2+
and Mn
2+
) destabilize the double helical structure. This is because all these ions exhibit an affinity to both phosphate and bases, with their association constants being significantly different, Cu
2+
for example has the highest affinity for DNA bases and in particular N-7 of guanine is the prime target for Cu
2+
complexation. Hence, when an increasing portion of the phosphata vacancies are filled, the affinity constant for the binding of a particular metal to phosphate decreases as a consequence of the cooperative binding nature. At this point, binding to the DNA bases becomes more important and competition for hydrogen bonding is initiated. The effect is manifested in a lowering of the melting temperature (T
m
) of the dsDNA (Eichhorn and Shin, J.Am.Chem.Soc., 90: 7323 (1968) P -Y. Cheng, Biochem. Biophys.Acta., 102: 314 (1962); Schreiber and Daune, Biopolymers, 8: 130 (1969)).
The present invention provides alternative solutions for the above-mentioned problem. Instead of providing thermostabile DNA polymerases to cope with the extremely high temperatures needed to allow the DNA to denature, the present invention provides means for manipulating the conditions such that the DNA can denature at much lower temperatures and consequently no longer draws upon the use of thermostabile DNA polymerases.
It is thus an aim of the present invention to provide an alternative PCR amplification process.
It is also an aim of the present invention to provide an alternative PCR amplification process, that does not draw upon the use of thermostabile DNA polymerases.
It is also an aim of the present invention to provide an alternative type of PCR amplification kits.
It is further an aim of the present invention to provide an alternative type of PCR amplification device.
According to a preferred embodiment, the present invention relates to the use of a controlled oscillation of the concentration of divalent metal ions such as Cu
2+
, Cd
2+
, Zn
2+
and Mn
2+
, thereby forming the basis for isothermal denaturation of the double helix.
The present invention also provides means for the automatization of this process. For instance, the new method favours dynamic electrochemical control of the activity of ionic species present. Furthermore only divalent metal ions are considered at this stage. This however, by no means excludes the potential to extend electrolytic control to the activity of mono-valent cations, which are equally important members in terms of contributions to total ionic strength. The present invention departs from methods that allow separation of both strands of a DNA helix. This is not achieved solely by increasing the temperature but also by increasing the local concentration of divalent metal ions that have the tendency to destabilize the DNA helix. Destabilization of the DNA helix is reflected in the lowering of the melting temperature (T
m
) of DNA (i.e. the midpoint in the transition of dsDNA to ssDNA). Cu
2+
ions stabilize the double helical structure at low concentrations. However, at elevated concentrations Cu
2+
ions start to interfere with hydrogen bonding resulting in the transition from double helical into ssDNA.
According to a preferred embodiment the present invention relates to a process for amplifying at least part of a specific double-stranded nucleic acid sequence contained in a sample comprising:
(a) separating the nucleic acid strands in said sample essentially with a means for increasing the local concentration of metal ions, preferably of divalent metal ions;
(b) treating the strands with at least one oligonucleotide primer under hybridizing conditions essentially with a means for decreasing the local concentration of metal ions, preferably divalent metal ions, and in the presence of an inducing agent for polymerization and the different nucleotides, such that an extension product of the respective primer(s) is synthesized which is complementary to one end of the sequence to be amplified on one of the strands such that an extension product can be synthesized from said primer which, when it is separated from its complement, can serve as a template for synthesis of an extension product of the other primer;
(c) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules essentially with a means for increasing the local concentration of metal ions, preferably divalent metal ions;
(d) treating the single-stranded molecules generated from step (c) with the primers of step (b) under hybridizing conditions essentially with a means for decreasing the local concentration of metal ions, preferably divalent metal ions, and in the presence of an inducing agent for polymerisation and the different nucleotides such that a primer extension product is synthesized using each of the single-strands produced in step (c) as a template; and, if desired;
(e) repeating steps (c) and (d) at least once; whereby the amount of the sequence to be amplified increases exponentially relative to the number of ste
Rossau Rudi
Wijnhoven Michael
Horlick Kenneth R.
Innogenetics N.V.
Muserlian Lucas and Mercanti
Spiegler Alexander H.
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