ISOTHERMAL POLYMERASE CHAIN REACTION BY CYCLING THE CONCENTRATION OF DIVALENT METAL IONS

摘要

The present invention provides an alternative PCR amplification which does not draw upon the use of thermostable DNA polymerases. It provides means for the controlled manipulation of denaturing conditions which do not demand the use of high denaturing temperature. More particularly, it provides means for the controlled oscillation of divalent metal ions, preferably of divalent metal ions such as Cu2+, Zn2+, Mn2+ and Cd2+, which are known to destabilize the DNA helix and thereby decrease the melting temperature of the DNA helix. The invention also provides methods for the automatization of this process. For instance, by means of cathodic reduction of the divalent metal species the concentration can be decreased to levels that allows for reannealing of separated strands with the primers; while oxidation of deposited metals or oxidation of monovalent metal ions, can restore the initially high concentration that allows for separation of both strands that make up the DNA helix. Electrolytic control of metal ion activity hence provides a tool for the repetitive isothermal denaturation of duplex DNA, and consequently can be used as a substitute for thermal cycling in the amplification of genetic material. Isothermal denaturation of dsDNA may be of considerable importance in the biotechnology and biomedical industry. A key advantage of this method is that it opens perspectives for a wide range of DNA polymerases that can be used with this reaction.