Endonuclase-assisted isothermal amplification using contamination-free reagents

Abstract

Disclosed are methods and kits for endonuclease-assisted DNA amplification reaction using decontaminated primer solutions that are pre-treated with a nuclease. Nucleic acid amplification assays that employ nuclease-resistant, inosine-containing primers, endonuclease V enzymes to introduce a nick into a target DNA comprising at least one inosine, and a DNA polymerase to generate amplicons of a target DNA are also disclosed.

1 . A method of producing at least one amplicon based on a target DNA comprising:
 (a) providing the target DNA;   (b) providing a primer solution comprising at least one exonuclease-resistant, inosine-containing primer;   (c) generating a DNA amplification reaction mixture by mixing together the target DNA, the primer solution, at least one 5′→3′ exonuclease-deficient DNA polymerase having a strand displacement activity, and at least one endonuclease that is capable of nicking an inosine-containing strand of a double stranded DNA at a residue 3′ to an inosine residue; and   (c) incubating the DNA amplification reaction mixture to amplify at least one portion of the target DNA using the at least one exonuclease-resistant, inosine-containing primer to produce the at least one amplicon.   
     
     
         2 . The method  claim 1 , wherein the primer solution further comprises free deoxynucleotide triphosphates (dNTPs). 
     
     
         3 . The method of  claim 2 , wherein the primer solution further comprises DNA amplification buffer, formamide, single stranded DNA binding protein, ethylene glycol, Ficoll or combinations thereof. 
     
     
         4 . The method of  claim 1 ,  claim 2 , or  claim 3  further comprising decontaminating the primer solution, prior to generating the DNA amplification reaction mixture, by treating the primer solution with an exonuclease to remove any contaminating nucleic acids. 
     
     
         5 . The method of  claim 4 , further comprising inactivating the exonuclease in the primer solution after removal of any contaminating nucleic acids prior to generating the DNA amplification reaction mixture. 
     
     
         6 . The method of  claim 5 , wherein the target DNA is amplified under isothermal conditions. 
     
     
         7 . The method of  claim 1 , wherein the inosine residue of the nuclease-resistant, inosine-containing primer is located at least 4 nucleotides downstream of the 5′ terminal nucleotide. 
     
     
         8 . The method of  claim 7 , wherein the inosine residue of the nuclease-resistant, inosine-containing primer is located at the penultimate 3′ position. 
     
     
         9 . The method of  claim 8 , wherein the nuclease-resistant, inosine-containing primer comprises a phosphorothioate linkage at the 5′ side or at the 3′ side of the inosine residue. 
     
     
         10 . The method of  claim 1 , wherein the inosine-containing primer comprises at least 2 adjacent inosine residues. 
     
     
         11 . The method of  claim 10 , wherein the inosine residues are located both at the penultimate 3′ position and the 3′ terminal end of the inosine-containing primer. 
     
     
         12 . The method of  claim 1 , wherein the at least one 5′→3′ exonuclease-deficient DNA polymerase is selected from 5′→3′ exonuclease-deficient T7 DNA polymerase, 5′→3′ exonuclease-deficient Bst DNA polymerase, 5′→3′ exonuclease-deficient Klenow, 5′→3′ exonuclease-deficient delta Tts DNA polymerase, or combinations thereof. 
     
     
         13 . The method of  claim 1 , wherein the primer solution comprises at least one inosine-containing forward primer and at least one inosine-containing reverse primer, wherein both the forward primer and the reverse primer are exonuclease-resistant. 
     
     
         14 . The method of  claim 1 , wherein the at least one nuclease-resistant, inosine-containing primer comprises an extender template. 
     
     
         15 . A method for producing at least one amplicon based on a target DNA comprising:
 (a) providing the target DNA;   (b) providing a primer solution consisting essentially of an exonuclease-resistant, inosine-containing primer;   (c) treating the primer solution with an exonuclease to remove any contaminating nucleic acids from the primer solution;   (d) inactivating the exonculease in the primer solution after step (b).   (e) generating a DNA amplification reaction mixture by mixing together the target DNA, the primer solution of step (d), a 5′→3′ exonuclease-deficient DNA polymerase having strand displacement activity, and a endonuclease that is capable of nicking a DNA at a residue 3′ to an inosine residue; and   (f) amplifying at least one portion of the target DNA using the amplification reaction mixture of step (e) to produce the at least one amplicon.   
     
     
         16 . The method  claim 15 , wherein the primer solution further comprises free nucleotides (dNTPs) and reagents slected from the group consisting of DNA amplification buffer, formamide, single stranded DNA binding protein, ethylene glycol, Ficoll and combinations thereof. 
     
     
         17 . A kit for endonuclease-assisted isothermal nucleic acid amplification, comprising:
 at least one exonuclease-resistant, inosine-containing primer;   at least one 5′→3′ exonuclease-deficient DNA polymerase with strand displacement activity; and   at least one endonuclease, which is capable of nicking DNA at a residue 3′ to an inosine residue.   
     
     
         18 . The kit of  claim 17 , wherein the inosine residue of the inosine-containing primer is located at least 4 nucleotides downstream of the 5′ terminal nucleotide. 
     
     
         19 . The kit of  claim 18 , wherein the inosine residue of the inosine-containing primer is located at the penultimate 3′ position. 
     
     
         20 . The kit of  claim 19 , wherein the inosine-containing primer comprises at least one phosphorothioate linkage between the inosine residue at the penultimate 3′ position and the 3′ terminal residue. 
     
     
         21 . The kit of  claim 20 , wherein the at least one 5′→3′ exonuclease-deficient DNA polymerase is selected from 5′→3′ exonuclease-deficient T7 DNA polymerase, 5′→3′ exonuclease-deficient Bst DNA polymerase, 5′→3′ exonuclease-deficient Klenow, 5′→3′ exonuclease-deficient delta Tts DNA polymerase, or combinations thereof. 
     
     
         22 . The kit of  claim 21 , wherein the at least one nuclease is an endonuclease V.