Isothermal polymerase chain reaction by cycling the concentration of divalent metal ions

Abstract

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 Cu 2+ , Zn 2+ , Mn 2+ and Cd 2+ , 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.

1 . 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 or 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 steps in which primer extension products are synthesized.    
     
     
         2 . A process according to    claim 1    wherein said metal ion is at least one of the following divalent metal ions: Cu2+ and/or Cd 2+  and/or Zn 2+  and/or Mn 2+  with said metal ion of    claim 1    consisting either of one kind of metal ions or of a mixture of different kinds of metal ions.  
     
     
         3 . A process according to any of claims  1  or  2  wherein said means for increasing the metal ion concentration comprises: 
 a means for adding said metal in a to increase its concentration, possibly through dialysis, or,  
 a means for oxidizing said metal to the monovalent form and/or a divalent form and/or oxidizing the monovalent metal form to the divalent form,  
 with said means possibly also comprising:  
 an agent which can assure the reversibility of said nucleic acid strand separation process, or,  
 a procedure that can increase, the nucleic acid strand separating effect of said divalent metal ion concentration increase, comprising the lowering of the apparent concentration of electrolytes present in the reaction mixture, preferably of Mg 2+  ions, and/or an increase of the reaction temperature.  
 
     
     
         4 . A process according to any of claims  1  or  2  wherein said means for decreasing the divalent metal ion concentration comprises: 
 a means for removing said divalent and/or monovalent metal ions, preferably through dialysis, or,  
 a means for reducing said divalent metal ions and/or monovalent metal ions, and,  
 with said means possibly also comprising:  
 an agent which can assure or facilitate the reversibility of said nucleic acid strand annealing process, or,  
 a procedure that can increase the nucleic acid strand annealing effect of said divalent metal ion concentration decrease, comprising an increase of the apparent concentration of electrolytes present in the reaction mixture, preferably of Mg 2+  ions, and/or a lowering of the reaction temperature.  
 
     
     
         5 . A process for detecting and/or purifying 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, whereby the extension products can be kept very short;    and optionally, followed by steps (c) and (d)    (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 primer or primers of step (b) under hybridizing conditions essentially with a means for decreasing the local concentration of metal ions, preferably divalent metal ions,; and, if desired;    (e) repeating steps (c) and (d) at least once; whereby the amount of the sequence to be amplified increases.    
     
     
         6 . A process for detecting and/or purifying 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) contacting the strands with at least one nucleic acid, preferably a primer or a set of primers under hybridizing conditions essentially with a means for decreasing the local concentration of metal ions, preferably divalent metal ions;    (c) separating the primers from the templates on which they annealed 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 primer or primers of step (b) under hybridizing conditions essentially with a means for decreasing the local concentration of metal ions, preferably divalent metal ions,; and, if desired;    (e) repeating steps (c) and (d) at least once; whereby detection and/or purification can be enhanced further.    
     
     
         7 . A process according to any of    claims 1    to    6    further characterized in that said amplified nucleic acid sequence is detected.  
     
     
         8 . A kit for the amplification and possible detection of at least part of a specific nucleic acid sequence contained in a sample comprising in a packaged form, a multi container unit comprising: 
 (a) a primer according to    claim 1   ;    (b) a means for synthesizing the primer extension products according to any of    claims 1    to    7   ; and possibly    (c) means for detecting the amplified sequence or sequences.    
     
     
         9 . A device for performing a process according to any of    claims 1    to    5    comprising a system that allows for the metal ion concentration and/or metal ion chelators to cycle in a controllable way, comprising for instance: 
 an electrode system that allows for reduction of divalent metal ions, preferably allows for a selective reduction of said metal ions, thereby creating the reaction conditions such that step (b) and/or (d) of    claim 1    can take place, and/or  
 an electrode system that allows for oxidation of metals or monovalent metal ions towards said divalent metal ions, preferably allows for a selective oxidation of said metals or monovalent metal ions, thereby restoring the reaction conditions such that step (a) and/or (c) of    claim 1    can take place, and/or  
 a system that allows for the cyclic addition of said divalent metal ions, thereby restoring the reaction conditions such that step (a) and/or (c) of    claim 1    can take place, and/or  
 a system that allows for the reaction components to stay contained in a vial separated from a surrounding medium through a dialysis membrane, and wherein the surrounding medium can be flushed in a cyclic and controlled way with solution 1, said flushing allowing for the cyclic increase of said metal ions and/or increase of the hydrophobicity of the reaction medium and/or lowering of the electrolyte concentration, preferably of Mg 2+  ions, followed by flushing in a cyclic and controlled way with solution 2, said flushing allowing for the cyclic decrease of said metal ions, possibly in combination with the use of metal chelators and/or decrease of the hydrophobicity of the reaction medium and/or increase of the electrolyte concentration, preferably of Mg 2+  ions, and/or  
 a system that allows for a fine tuned control of the reaction temperature.