Enzyme Composition for DNA End Repair, Adenylation, Phosphorylation

Abstract

Enzyme compositions and their method of use that provide ready-to-use master mixtures. The compositions comprise a modified thermophilic DNA polymerase lacking 5′-3′ and 3′-5′ exonuclease activity premixed with T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase and all other necessary components, including reaction buffer and nucleoside triphosphates, required to perform DNA blunting, phosphorylation, and single nucleotide extension reactions in one tube and in two steps. Among other benefits, the mixture of different enzymes, buffers and nucleoside triphosphates is stable during prolonged storage.

1 - 20 . (canceled) 
     
     
         21 . An enzyme composition comprising in a single container a buffer comprising nucleoside triphosphates and a plurality of enzymes separately capable of blunting, phosphorylating and adenylating DNA fragments, with the enzyme capable of adenylating DNA being a thermophilic modified DNA polymerase, and the composition being stable for at least one week at 4° C. 
     
     
         22 . The composition of  claim 21 , where the enzymes capable of blunting and phosphorylating DNA fragments are chosen from T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, and T4 polynucleotide kinase. 
     
     
         23 . The composition of  claim 21 , where the thermophilic modified DNA polymerase is a  Thermus  species thermophilic DNA polymerase fused to non-specific DNA-binding domain. 
     
     
         24 . The composition of  claim 21 , where the thermophilic modified DNA polymerase lacks 5′-3′ and 3′-5′ exonuclease activity. 
     
     
         25 . The composition of  claim 23 , where the thermophilic modified DNA polymerase is a  Thermus  species polymerase fused to non-specific DNA-binding domain from  Sulfolobus  family. 
     
     
         26 . The composition of  claim 21 , where the nucleoside triphosphates are dATP, dCTP, dTTP, and dGTP, where the concentration of dATP ranges from 0.4 to 3 mM, the concentration of dCTP ranges from 0.2 to 0.6 mM, the concentration of dTTP ranges from 0.2 to 0.6 mM, and the concentration of dGTP ranges from 0.1 to 0.4 mM. 
     
     
         27 . The composition of  claim 21 , where the concentration of dATP is about 2 mM, the concentration of dCTP is about 0.4 mM, the concentration of dTTP is about 0.4 mM, and the concentration of dGTP is about 0.2 mM. 
     
     
         28 . The composition of  claim 21 , where the buffer further comprises at least one component selected from the group consisting of Tris-HCl, MgCl 2 , DTT, a monovalent metal hydrochloric acid salt selected from NaCl, KCl, and LiCl, ATP, Triton X-100, glycerol, NP 40, EDTA, and Tween 20. 
     
     
         29 . The composition of  claim 27 , where, when present, the concentration of Tris-HCl ranges from 100 mM to 105 mM at a pH of 8.0 to 8.8; the concentration of MgCl 2  ranges from 15 to 25 mM, the concentration of DTT ranges from 15 to 30 mM, the concentration of monovalent metal hydrochloric acid salt ranges from 20 mM to 50 mM, the concentration of ATP ranges from 1.5 to 2.5 mM, the concentration of Triton X-100 ranges from 0.1 to 0.4%, the concentration of glycerol ranges from 10 to 20%, the concentration of NP 40 ranges from 0.05 to 0.15%, the concentration of EDTA ranges from 0.02 to 0.1 mM, and the concentration of Tween 20 ranges from 0.05 to 0.15%. 
     
     
         30 . The composition of  claim 22 , where the concentration of T4 DNA polymerase ranges from 0.2 to 0.5 U/μl, the concentration of Klenow fragment ranges from 0.1 to 0.2 U/μl, and the concentration of T4 polynucleotide kinase ranges from 0.5 U/μl to 1.5 U/μl. 
     
     
         31 . The composition of  claim 30 , where the concentration of T4 DNA polymerase is about 0.32 U/μl, the concentration of Klenow fragment is about 0.12 U/μl, and the concentration of T4 polynucleotide kinase is about 1 U/μl. 
     
     
         32 . The composition of  claim 23 , where the concentration of the thermophilic modified DNA polymerase ranges from 0.1 to 0.5 U/μl. 
     
     
         33 . The composition of  claim 23 , where the concentration of the thermophilic modified DNA polymerase is about 0.2 U/μl. 
     
     
         34 . An enzyme composition comprising in a single container Tris-HCl, MgCl 2 , DTT, KCl, dATP, dCTP, dTTP, dGTP, ATP, Triton X-100, glycerol, NP 40, EDTA, Tween 20, T4 polynucleotide kinase, T4 DNA polymerase, Klenow fragment, and thermophilic modified DNA polymerase from  Thermus brockianus  or  Thermus  sp. fused to DNA-binding domain from  Sulfolobus  family, the composition contained in a single container and being storage stable. 
     
     
         35 . The composition of  claim 34 , where the concentration of dATP is at least five times the concentration of each of dCTP, dTTP, and dGTP. 
     
     
         36 . A method for generating a DNA library, the DNA library comprising DNA fragments each having 3′ end and a 5′ end, the DNA fragments having synthetic DNA adapters joined to each of the 3′ and 5′ of the DNA fragments, the method comprising
 shearing a DNA sample to generate DNA fragments, 
 mixing the DNA fragments with the composition of claim  1  in a single container, 
 performing DNA end-repair and dA-tailing of the DNA fragments in the single container by 
 subjecting the contents of the single container to a first reaction condition promoting the DNA end-repair reaction, and thereafter 
 subjecting the contents of the single container to a second reaction condition promoting the dA-tailing reaction and inactivating the DNA end-repair reaction, 
 where the DNA end-repair reaction comprises blunting and phosphorylating the DNA fragments, and the dA-tailing reaction comprises adenylating the DNA fragments by adding a single dA to the 3′ ends of the end-repaired DNA fragments, and 
 ligating the end-repaired and dA-tailed DNA fragments to synthetic DNA adapters having dT extensions at the 3′ ends. 
 
     
     
         37 . The method of  claim 36 , where the first reaction condition comprises subjecting the container contents to a temperature of from 18° C. to 22° C. for a period of from 5 to 10 minutes, and the second reaction condition comprises subjecting the container contents to a temperature of from 72° C. to 75° C. for a period of from 10 to 20 minutes. 
     
     
         38 . The method of  claim 37 , where the first reaction condition comprises subjecting the container contents to a temperature of about 20° C. for about five minutes, and the second reaction condition comprises subjecting the container contents to a temperature of 72° C. for about ten minutes. 
     
     
         39 . The method of  claim 36 , where the yield of dA-tailed DNA fragments exceeds 75%. 
     
     
         40 . A kit comprising the composition of  claim 21  and instructions for performing the method of  claim 36 .