Contamination-free reagents for nucleic acid amplification

Abstract

Methods and kits for generating contamination-free reagents and reagent solutions for use in nucleic acid amplification are provided. Methods include processing of polymerase solutions, nucleotide solutions and primer solutions to render contaminating nucleic acid inert. The methods employ the proofreading activity of the polymerase and/or exonucleases to de-contaminate the reagents and reagent solutions. Methods and kits for contamination-free nucleic acid amplification are provided.

1 . A kit for DNA amplification comprising:
 (a) a proofreading DNA polymerase;   (b) a nuclease resistant primer; and   (c) an exonuclease.   
     
     
         2 . The kit of  claim 1 , further comprising a single stranded DNA-binding protein. 
     
     
         3 . The kit of  claim 1 , wherein the proofreading DNA polymerase comprises a Phi29 DNA polymerase. 
     
     
         4 . The kit of  claim 1 , wherein the exonuclease is selected from the group consisting of exonuclease I, exonuclease III and combinations thereof. 
     
     
         5 . The kit of  claim 1 , wherein the proofreading DNA polymerase is a decontaminated proofreading DNA polymerase. 
     
     
         6 . The kit of  claim 5 , wherein the decontaminated proofreading DNA polymerase is generated by a method comprising the steps of:
 (a) providing a polymerase solution consisting essentially of a proofreading DNA polymerase that is contaminated with a contaminating nucleic acid;   (b) contacting the polymerase solution with a divalent cation to form a polymerase-cation mixture; and   (c) incubating the polymerase-cation mixture whereby the contaminating nucleic acid is rendered inert by the proofreading DNA polymerase to generate the decontaminated proofreading DNA polymerase,   wherein the contacting and the incubating steps are performed in the absence of any substantial amount of free nucleotides (dNTPs), and wherein the decontamination is performed in absence of an exonuclease or DNAse.   
     
     
         7 . The kit of  claim 1 , wherein the nuclease-resistant primer is a decontaminated nuclease-resistant primer, and wherein the decontaminated nuclease-resistant primer is generated by a method comprising the steps of:
 (a) providing a contaminated nuclease-resistant primer solution that consists of a nuclease-resistant primer and a contaminating nucleic acid;   (b) contacting the primer solution with a nuclease and a divalent cation; and   (c) incubating the primer solution whereby the contaminating nucleic acid is rendered inert by the nuclease to generate the decontaminated nuclease-resistant primer.   
     
     
         8 . The kit of  claim 1 , wherein the nuclease-resistant primer is an exonuclease-resistant primer. 
     
     
         9 . The kit of  claim 1 , further comprising decontaminated free nucleotides (dNTPs), wherein the decontaminated free nucleotides (dNTPs) is generated by:
 (a) contacting a nucleotide solution with a nuclease and a divalent cation, wherein the nucleotide solution comprises free nucleotides and a contaminating nucleic acid; and   (b) incubating the nucleotide solution to allow the nuclease to render the contaminating nucleic acid inert.   
     
     
         10 . The kit of  claim 1 , further comprising a non-proofreading DNA polymerase. 
     
     
         11 . A kit for DNA amplification comprising:
 a decontaminated proofreading DNA polymerase, which is generated by a method comprising the steps of:   (a) providing a polymerase solution consisting essentially of a proofreading DNA polymerase that is contaminated with a contaminating nucleic acid;   (b) contacting the polymerase solution with a divalent cation to form a polymerase-cation mixture; and   (c) incubating the polymerase-cation mixture whereby the contaminating nucleic acid is rendered inert by the proofreading DNA polymerase to generate the decontaminated proofreading DNA polymerase,   wherein the contacting and the incubating steps are performed in the absence of any substantial amount of free nucleotides (dNTPs), and wherein the decontamination is performed in absence of an exonuclease or DNAse.   
     
     
         12 . The kit of  claim 11 , wherein the decontaminated proofreading DNA polymerase is a decontaminated Phi29 DNA polymerase. 
     
     
         13 . The kit of  claim 11 , further comprising a decontaminated non-proofreading DNA polymerase. 
     
     
         14 . A decontaminated proofreading DNA polymerase made by incubating a proofreading DNA polymerase with a divalent cation in the absence of any substantial amount of free nucleotides, wherein the incubation is performed in the absence of any exonuclease or DNAse. 
     
     
         15 . The decontaminated proofreading DNA polymerase of  claim 14 , wherein the polymerase is a Phi29 DNA polymerase.