Method and compositions for reversible inhibition of thermostable polymerases

Abstract

Methods for improving sensitivity and specificity of polynucleotide synthesis are disclosed. The method includes reversibly blocking thermophilic polymerase activity with non-nucleic acid polyanions in a temperature dependent manner. The methods control target specific primer extension throughout all stages of a DNA or RNA amplification reaction. Corresponding compositions and kits are disclosed.

What is claimed is:  
     
         1 . A method of polynucleotide synthesis, comprising: 
 combining in a polymerization reaction mixture a thermostable polymerase, a template nucleic acid molecule, appropriate primers for the template nucleic acid molecule, at least one deoxynucleoside triphosphate, and a non-nucleic acid polyanion, wherein the temperature of the polymerization reaction mixture is at a temperature at which the non-nucleic acid polyanion inhibitstermostable polymerase activity; and    heating the polymerization reaction mixture to a temperature at which the nonnucleic acid polyanion dissociates from the thermostable polymerase, allowing the thermostable polymerase to recognize and provide polynucleotide synthesis on a primer annealed nucleic acid molecule.    
     
     
         2 . The method of  claim 1  wherein the polynucleotide synthesis is polymerase chain reaction  
     
     
         3 . The method of  claim 1  wherein the non-nucleic acid polyanion has a molecular weight of from 1500 to 500,000.  
     
     
         4 . The method of  claim 1  wherein the non-nucleic acid polyanion has a molecular weight of from 4,000 to 15,000.  
     
     
         5 . The method of  claim 1  wherein the non-nucleic acid polyanion has a molecular weight of from 5,000 to 10,000.  
     
     
         6 . The method of  claim 1  wherein the non-nucleic-acid polyanion is a synthetic organic polysulfate selected from the group poly(anetholsulfonic acid) polyvinyl sulfate and polystyrene sulfate.  
     
     
         7 . The method of  claim 6  wherein the non-nucleic acid polyanion is a sulfated oligo- or polysaccharide.  
     
     
         8 . A method of polynucleotide synthesis, comprising: 
 combining in a polymerization reaction mixture a thermostable polymerase, a template nucleic acid molecule, appropriate primers for the template nucleic acid molecule, at least one deoxynucleoside triphosphate, and a polymer or copolymer of sugars selected from the group consisting of glucose, N-acetyl-glucosamine, galactouronic acid, hyalouronic acid, Nacetyl-galactosamine and sulfated fucose, wherein the temperature of the polymerization reaction mixture is at a temperature at which the polymer or copolymer inhibits thermostable polymerase activity;    heating the polymerization mixture to a temperature at which the template nucleic acid molecule is denatured from a double-stranded molecule to a single-stranded molecule;    cooling the polymerization mixture to a temperature of from about 45° C. to about 65° C. to allow appropriate primers to anneal to the single-stranded molecule; and    modifying the polymerization mixture to a temperature at which the polymer or copolymer is substantially dissociated from the thermostable polymerase and the thermostable polymerase recognizes and provides polynucleotide synthesis on primer annealed nucleic acid molecule.    
     
     
         9 . The method of  claim 8  wherein the sulfated polymer or copolymer of sugars is selected from the group consisting of dextran sulfate, fucoidan, heparin; heparan sulfate, chondroitin polysulfate, keratan polysulfate, xylaR poly, sulfate, and pentosan polysulfate.  
     
     
         10 . The method of  claim 1  wherein the non-nucleic acid polyanion is at a final reaction concentration of from 0.1 μM to 1.5 μM.  
     
     
         11 . The method of  claim 1  wherein the non-nucleic acid polyanion is at a final reaction concentration of from 0.2 μM to 1.0 μM.  
     
     
         12 . A method of polynucleotide synthesis, comprising: 
 combining in a polymerization reaction mixture a thermostable polymerase, a template nucleic acid molecule, appropriate primers for the template nucleic acid molecule, at least one deoxynucleoside triphosphate, and a non-nucleic acid polyanion, wherein the temperature of the polymerization reaction mixture is at a temperature at which the non-nucleic acid polyanion inhibits thermostable polymerase activity;    heating the polymerization reaction mixture to a temperature at which the template nucleic acid molecule is denatured from a double-stranded molecule to a single-stranded molecule;    cooling the polymerization reaction mixture to a temperature at which appropriate primers anneal to the single-stranded molecule; and    modifying the temperature of the polymerization reaction mixture to 60° C. to 75° C. wherein the non-nucleic polyanion substantially ceases to inhibit thermostable polymerase activity.    
     
     
         13 . A method of polynucleotide synthesis, comprising: 
 combining in a polymerization reaction mixture a thermostable polymerase selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, and mixtures thereof, a template nucleic acid molecule, appropriate primers for the template nucleic acid molecule, at least one deoxynucleoside triphosphate, and a non-nucleic acid polyanion, wherein the temperature of the polymerization reaction mixture is at a temperature at which the non-nucleic acid polyanion inhibits thermostable polymerase activity;    heating the polymerization reaction mixture to a temperature at which the template nucleic acid molecule is denatured from a double-stranded molecule to a single-stranded molecule;    cooling the polymerization reaction mixture to a temperature at which appropriate primers anneal to the single-stranded molecule; and    modifying the temperature of the polymerization reaction mixture to a temperature at which the non-nucleic polyanion is substantially dissociated from the thermostable polymerise, wherein the thermostable polymerise recognizes and provides polynucleotide synthesis on primer annealed nucleic acid molecule.    
     
     
         14 . The method of  claim 13  wherein the reverse transcriptase is a derivative, mutant or chimeric complex of the reverse transcriptase.  
     
     
         15 . A kit for polynucleotide synthesis on a target nucleic acid, the kit comprising: 
 a thermostable polymerase reversibly bound to a non-nucleic acid polyanion; and    an appropriate polymerase reaction buffer.    
     
     
         16 . The kit of  claim 15  wherein the thermostable polymerase is  Thermus aquaticus.    
     
     
         17 . The kit of  claim 15  wherein the non-nucleic acid polyanion is dextran sulfate.  
     
     
         18 . The kit of  claim 15  further comprising at least one nucleotide 5′-triphosphate.  
     
     
         19 . The kit of  claim 15  further comprising a pair of primers for the target nucleic acid.  
     
     
         20 . The kit of  claim 15  wherein the non-nucleic acid polyanion has a molecular weight of from 1,500 to 500,000.  
     
     
         21 . The kit of  claim 15  wherein the non-nucleic acid polyanion has a molecular of from 4,000 to 15,000.  
     
     
         22 . A composition for polynucleotide synthesis comprising: 
 a thermostable polymerase;    a non-nucleic acid polyanion;    a polymerase reaction buffer having monovalent cations between 35-60 mM;    at least one dNTP;    a template nucleic acid molecule;    and appropriate template nucleic acid primers.    
     
     
         23 . The composition of  claim 22  wherein the non-nucleic acid polyanion has a molecular weight of from 1,500 to 500,000.  
     
     
         24 . The composition of  claim 22  wherein the non-nucleic acid polyanion has a molecular weight of from 4,000 to 15,000.  
     
     
         25 . The composition of  claim 22  wherein the non-nucleic acid polyanion has a molecular weight of from 4,000 to 10,000.  
     
     
         26 . The composition of  claim 22  wherein the non-nucleic acid polyanion is a synthetic organic polysulfate selected from the group poly(anetholsulfonic acid), polyvinyl sulfate, and 15 polystyrene sulfate.  
     
     
         27 . The composition of  claim 26  wherein the anionic polysulfate is a sulfated oligo- or polysaccharide.  
     
     
         28 . The composition of  claim 27  wherein the sulfated oligo- or polysaccharide is a sulfated polymer or copolymer of the sugars selected from the group consisting essentially of glucose, N-acetyl-glucosamine, galactouronic acid, hyalouronic acid, N-acetyl-galactosamine and fucose.  
     
     
         29 . The composition of  claim 28  wherein the sulfated polymer or copolymer of the sugar is selected from the group consisting essentially of dextran sulfate, fucoidan, heparin, heparan sulfate, chondroitin polysulfate, keratan polysulfate, xylan polysulfate, and pentosan polysulfate.  
     
     
         30 . The composition of  claim 22  wherein the non-nucleic acid polyanion is at a concentration of from 0.1 μM to 1.5 μM.  
     
     
         31 . The composition of  claim 22  wherein the non-nucleic acid polyanion is at a concentration of from 0.2 μM to 1.0 .μM.  
     
     
         32 . The composition of  claim 22  wherein the thermostable polymerase is selected from the group consisting essentially of DNA polymerase, RNA polymerase, reverse transcriptase, and mixtures thereof.  
     
     
         33 . The composition of  claim 32  wherein the thermostable polymerase is a DNA polymerase and the DNA polymerase is from a thermophilic Eubacteria or a Archaebacteria.  
     
     
         34 . The composition of  claim 33  wherein the thermostable polymerase is selected from the group consisting essentially of  Thermus aquaticus, T. thermophilus, T. brockianus, T. flavus, T. ruber, Thermatoga maritima, Thermoplasma acidophilus, Pyroccocus furiosus, Pyroccocus woesii , Pyroccocus spec., Sulfolobus spec., and mixtures thereof.  
     
     
         35 . The composition of  claim 32  wherein the thermostable polymerase is a reverse transcriptase and wherein the reverse transcriptase is selected from the group consisting essentially of MmLV reverse transcriptase, AMV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, and mixtures thereof.  
     
     
         36 . The method of  claim 12  wherein the non-nucleic acid polyanion has a molecular weight of from 4,000 to 15,000.  
     
     
         37 . The method of  claim 12  wherein the non-nucleic acid polyanion has a molecular weight of from 4,000 to 10,000.  
     
     
         38 . The method of  claim 8  wherein the modifying of the polymerization mixture to a temperature at which the non-nucleic polyanion is substantially dissociated from the thermostable polymerase is from 60° C. to 75° C.