Polymerase

Abstract

The present invention relates to an engineered polymerase characterized in that the polymerase exhibits an enhanced ability to process nucleic acid in the presence of environmental and biological inhibitors compared to wild type DNA polymerase.

1 . An engineered polymerase characterised in that it exhibits an enhanced ability to process nucleic acid (i) in the presence of humic acid; (ii) in the presence of one or more phenolic compounds or derivatives thereof; or (iii) in the presence of soil,
 compared to wild type polymerase.   
     
     
         2 - 5 . (canceled) 
     
     
         6 . The engineered polymerase according to  claim 1  which exhibits an enhanced ability to process nucleic acid in the presence of (i) humic acid; phenolic acid or derivatives thereof; or phytophenolic acid or derivatives thereof,
 at a concentration of between 5 and 20%.   
     
     
         7 - 8 . (canceled) 
     
     
         9 . The engineered polymerase according to  claim 1  wherein the engineered polymerase is derived from wild type polymerase by substitution, deletion or insertion of one or more amino acids. 
     
     
         10 . The engineered polymerase according to  claim 1  wherein said ability to process nucleic acid is enhanced at least four fold, eight fold, or sixteen fold, when compared to the wild type polymerase. 
     
     
         11 - 12 . (canceled) 
     
     
         13 . The engineered polymerase according to  claim 1  wherein the ability to process nucleic acid within a polymerase chain reaction is enhanced. 
     
     
         14 . The engineered polymerase according to  claim 1  comprising an engineered polymerase that is generated from a library derived by recombining related wild type polymerase genes. 
     
     
         15 . The engineered polymerase according to  claim 1  wherein said wild type polymerase is selected from a group consisting of Taq, T8, TTh and Ttl. 
     
     
         16 . The engineered polymerase according to  claim 1  wherein said polymerase is generated from a library of nucleic acids derived by error prone polymerase chain reaction mutagenesis and/or recombination of related wild type polymerase genes. 
     
     
         17 . The engineered polymerase according to  claim 1  wherein the polymerase is a DNA polymerase. 
     
     
         18 . A method for producing the engineered polymerase of  claim 1 , which comprises:
 (a) preparing a nucleic acid molecule encoding a polymerase;   (b) introducing a mutation into the nucleic acid molecule encoding that polymerase according to step (a) so that one or more nucleotides in one or more regions are not identical to the polymerase from which it is derived;   (c) selecting a modified polymerase expressed by the mutated nucleic acid molecule by the ability of said modified polymerase to process nucleic acid in the presence of (i) humic acid; (ii) one or more phenolic compounds or derivatives thereof; or (iii) soil; and   (d) isolating and purifying that polymerase.   
     
     
         19 - 21 . (canceled) 
     
     
         22 . A method for the generation of an engineered polymerase according to  claim 1  which comprises the steps of:
 (a) providing a pool of nucleic acids comprising members each encoding an engineered polymerase;   (b) providing (i) humic acid; (ii) one or more phenolic compounds or derivatives thereof; or (iii) soil;   (c) subdividing the pool of nucleic acids into compartments, such that each compartment comprises substantially a nucleic acid member of the pool together with the engineered polymerase encoded by the nucleic acid member, and (i) humic acid; (ii) one or more phenolic compounds or derivatives thereof; or (iii) soil;   (d) allowing processing of the nucleic acid member to occur; and   (e) detecting processing of the nucleic acid member by that engineered polymerase;   (f) optionally repeating the series of steps (a) to (f) one or more times; and   (g) isolating and purifying that engineered polymerase.   
     
     
         23 - 25 . (canceled) 
     
     
         26 . The method according to  claim 22  wherein humic acid is provided at a concentration that inhibits wild type polymerase activity. 
     
     
         27 . The method according to  claim 22  wherein one or more phenolic compounds are provided at a concentration that inhibits wild type polymerase activity. 
     
     
         28 - 32 . (canceled) 
     
     
         33 . An isolated nucleic acid molecule which encodes an engineered DNA polymerase polypeptide comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% identity to any of SEQ ID NOs 2, 4 or 6 and wherein said polypeptide has DNA polymerase activity in the presence of 5 to 20% humic acid. 
     
     
         34 - 36 . (canceled) 
     
     
         37 . An isolated nucleic acid molecule encoding an engineered DNA polymerase comprising a nucleotide sequence as set forth in any of SEQ ID NOs 1, 3, 5, 7 or 9, or a nucleotide sequence having at least 80%, 90%, or 95% sequence identity with any of SEQ ID NOs 1, 3, 5, 7, or 9, wherein said polymerase exhibits an enhanced ability to process nucleic acid in the presence of humic acid compared to wild type polymerase. 
     
     
         38 - 40 . (canceled) 
     
     
         41 . The engineered polymerase according to  claim 1  wherein said engineered polymerase comprises an amino acid sequence that has at least 80%, 90%, or 95% identity to amino residues of the wild type polymerase. 
     
     
         42 - 43 . (canceled) 
     
     
         44 . A polypeptide with DNA polymerase activity, characterized in that the amino acid sequence of that polypeptide comprises the amino acid sequence of any of SEQ ID NOs 2, 4, 6, 8 or 10. 
     
     
         45 - 46 . (canceled) 
     
     
         47 . A recombinant nucleic acid molecule comprising a promoter sequence operably linked to the nucleic acid molecule according to  claim 33 . 
     
     
         48 . A cell transformed with the recombinant nucleic acid molecule according to  claim 47 . 
     
     
         49 - 50 . (canceled) 
     
     
         51 . A kit for amplifying nucleic acid comprising an isolated, engineered polymerase according to  claim 1 .