DNA polymerase

Abstract

The present invention relates to DNA polymerases. In particular the invention relates to a method for the generation of DNA polymerases exhibiting a relaxed substrate specificity. Uses of mutant polymerases produced using the methods of the invention are also described.

1 . A method for the generation of an engineered DNA polymerase with an expanded substrate range which method comprises the step of preparing and expressing nucleic acid encoding an engineered DNA polymerase, wherein said preparing comprises the use of template DNA polymerase nucleic acid and primers which bear one or more distorting 3′ termini.  
     
     
         2 . A method for the generation of a engineered DNA polymerase with an expanded substrate range which comprises the steps of: 
 (a) preparing nucleic acid encoding an engineered DNA polymerase, wherein the polymerase is generated using a repertoire of nucleic acid molecules encoding one or more DNA polymerases and primers which bear distorting 3 termini.    (b) compartmentalising the nucleic acid of step (a) into microcapsules;    (c) expressing the nucleic acid to produce their respective DNA polymerase within the microcapsules;    (d) sorting the nucleic acid encoding the engineered DNA polymerase which exhibits an expanded substrate range; and    (e) expressing the engineered DNA polymerase which exhibits an expanded substrate range.    
     
     
         3 . The method of  claim 1  wherein the distorting 3′ terminus of the primers is effected by any one or more of the following techniques: the presence of one nucleotide mismatch bases at the 3′end of the one or more primers (primer mismatch disortion); the presence of two nucleotide mismatch bases at the 3′end of the one or more flanking primers (primer mismatch disortion); the presence of three nucleotide mismatch bases at the 3′end of the one or more flanking primers (primer mismatch disortion); the presence of four nucleotide mismatch bases at the 3′end of the one or more flanking primers (primer mismatch disortion) and the presence of one or more unnatural bases at the 3′ end of one or more flanking primers or combinations thereof.  
     
     
         4 . The method of  claim 3  wherein the distorting 3′ terminus of the primers is effected by the presence of 5nitroindole triphosphate (5NITP) at the 3′ end of one or more flanking primers.  
     
     
         5 . The method of  claim 4  wherein the flanking primers bear one or more nucleotide mismatches at their 3′ end.  
     
     
         6 . The method of  claim 1  wherein the engineered DNA polymerase is a pol A DNA polymerase.  
     
     
         7 . The method of  claim 6  wherein the pol A polymerase is generated from a repertoire of randomly mutated Taq genes.  
     
     
         8 . The method of  claim 6  wherein the pol A polymerase is generated from repertoires generated by recombining related pol A genes.  
     
     
         9 . The method of  claim 7  wherein the pol A polymerase is generated from a repertoire generated by recombining one or more polA genes selected from the group consisting of:  Thermus aquaticus  (Taq),  Thermus thermophilus  (Tth) and  Thermus flavus  (Tfl).  
     
     
         10 . The method of  claim 6  wherein the pol A DNA polymerase is generated from a repertoire of Taq genes and wherein the pol A DNA polymerase is generated using 3′ mismatch flanking primers selected from the group consisting of: 5′-CAG GAA ACA GCT ATG ACA AAA ATC TAG ATA ACG AGG GA-3′; (A∘G mismatch); and 5′GTA AAA CGA CGG CCA GTA CCA CCG AAC TGC GGG TGA CGC CAA GCC-3′ (C*C mismatch).  
     
     
         11 . The method of  claim 6  wherein the pol A polymerase is generated from a repertoire of Taq genes or from a repertoire of chimeras of Taq, Tth and Tfl genes, wherein the pol A DNA polymerase is generated using flanking primers with four mismatches at their 3′ end (underlined), said primers selected from the group consisting of: 5′-CAG GAA ACA GCT ATG ACA AAA GTG AAA TGA ATA GTT CGA C TTTT -3′; 5′-GTA AAA CGA CGG CCA GTC TTC ACA GGT CAA GCT TAT TAA  GGTG -3′; 5′-CAG GAA ACA GCT ATG ACC ATT GAT AGA GTT ATT TTA CCA C AGGG -3′; 5′-GTA AAA CGA CGG CCA GTC TTC ACA GGT CAA GCT TAT TAA  GGTG -3′ 
     
     
         12 . The method of  claim 6  wherein the pol A polymerase is generated from a repertoire of Taq genes or from a repertoire of chimeras of Taq, Tth and Tfl genes, wherein the pol A DNA polymerase is generated using flanking primers containing unnatural base analogues (X) at their 3′ end.  
     
     
         13 . The method of  claim 12  wherein said flanking primers are selected from the group consisting of: 5′-CAG GAA ACA GCT ATG ACA AAA ATC TAG ATA ACG AGG GCA X-3′ and 5′-GTA AAA CGA CGG CCA GTA CCA CCG AAC TGC GGG TGA CGC CAA GCX-3′.  
     
     
         14 . The method of  claim 12  wherein X is 5-nitroindole.  
     
     
         15 . The method of  claim 12  wherein a said flanking primer further comprises an internal unnatural base analog (X).  
     
     
         16 . The method of  claim 15  wherein said internal unnatural base analog is 5-nitroindole.  
     
     
         17 . The method of  claim 15  wherein said flanking primers are selected from the group consisting of: 5′-CAG GAA ACA GCT ATG ACA AAA ATC TAG ATA XCG AGG GCA X-3′ and 5′-GTA AAA CGA CGG CCA GTA CCA CXG AAC TGC GGG TGA CGC CAA GCX-3′.  
     
     
         18 . The method of  claim 1  which comprises the step of preparing and expressing nucleic acid encoding a blend of engineered polymerases.  
     
     
         19 . An engineered DNA polymerase which exhibits an expanded substrate range relative to the wild-type form of said DNA polymerase.  
     
     
         20 . An engineered DNA polymerase produced by the method of  claim 1 .  
     
     
         21 . A engineered DNA polymerase which exhibits an expanded substrate range produced by the method of  claim 12 .  
     
     
         22 . The engineered DNA polymerase of  claim 20  which is an engineered pol A polymerase.  
     
     
         23 . The engineered DNA polymerase of  claim 21  which is an engineered pol A polymerase.  
     
     
         24 . An isolated DNA polymerase as shown in  FIG. 1   b  or  2   b.    
     
     
         25 . An isolated nucleic acid encoding a DNA polymerase with expanded substrate range, said nucleic acid comprising the sequence shown in  FIG. 1   a  or  2   a.    
     
     
         26 . A pol A DNA polymerase with an expanded substrate range, wherein the polymerase exhibits at least 95% identity to one or more of the amino acid sequences designated M1 and M4 as shown in  FIG. 1  and  FIG. 2  respectively and depicted SEQ No 1 and SEQ No 2 respectively and which comprises any one or more of the following mutations: E520G, D144G, L254P, E520G, E524G, N583S, 1.1-D144G, L254P, E520G, E524G, N583S, V113I, A129V, L245R, E315K, G364D, G403R, E432D, P481A, I614M, R704W, D144G, G370D, E742G, K56E, I63T, K127R, M3171, Q680R, R343G, G370D, E520G, G12A, A109T, D251E, P387L, A608V, R617K, D655E, T710N, E742G, A109T, D144G, V155I, P298L, G370D, 1614M, E694K, R795G, E39K, R343G, G370D, E520G, T539A, M747V, K767R, G84A, D144G, K314R, E520G, F598L, A608V, E742G, D58G, R74P, A109T, L245R, R343G, G370D, E520G, N583S, E694K, A743P.  
     
     
         27 . A pol A DNA polymerase which is capable of mismatch extension, wherein the DNA polymerase comprises the amino acid sequence of any one or more of the clones designated herein as 3B5, 3B8, 3C12 and 3D1.  
     
     
         28 . The DNA polymerase of  claim 27  which consists of the amino acid sequence of a clone designted herein as 3B5, 3B8, 3C12 or 3D1.  
     
     
         29 . A pol A DNA polymerase which is capable of abasic site bypass, wherein the DNA polymerase comprises the amino acid sequence of any one of the clones designated herein as 3A10, 3B6 or 3B11.  
     
     
         30 . The DNA polymerase of  claim 29  which consists of the amino acid sequence of a clone designated herein as 3A10, 3B6 or 3B11.  
     
     
         31 . A pol A DNA polymerase which is capable of DNA replication involving the incorporation of un-natural base analogues into the newly replicated DNA, wherein the pol A DNA polymerase comprises the amino acid sequence of a clone designated herein as 4D11 or 5D4.  
     
     
         32 . The pol A DNA polymerase of  claim 31  which consists of the amino acid sequence of clone 4D11 or 5D4.  
     
     
         33 . A pol A DNA polymerase with an expanded substrate range, wherein the polymerase exhibits at least 95% identity to one or more of the amino acid sequences herein designated 3B5, 3B8, 3C12, 3D1, 3A10, 3B6, 3B11, 4D11 and 5D4 which comprises a mutation, relative to one of parent genes Taq, Tth, or Tfl, found in clones 3B5, 3B8, 3C12, 3D1, 3A10, 3B6, 3B11, 4D11 and 5D4 disclosed herein.  
     
     
         34 . A nucleic acid construct encoding an engineered polymerase of  claim 20 .  
     
     
         35 . A nucleic acid construct encoding an engineered pol A DNA polymerase which exhibits an expanded substrate range, wherein said pol A DNA polymerase is the polymerase shown in  FIG. 1   b  or  2   b.    
     
     
         36 . A vector comprising a nucleic acid construct of  claim 34 .  
     
     
         37 . A vector comprising a nucleic acid construct of  claim 35 .  
     
     
         38 . A method of producing a polynucleotide, the method comprising contacting a template nucleic acid with a DNA polymerase of  claim 20 .  
     
     
         39 . The method of  claim 38  wherein said method comprises a process selected from the group consisting of: PCR amplification, sequencing of damaged DNA templates, the incorporation of un-natural base analogues into DNA and the creation of novel polymerase activities.  
     
     
         40 . A method of producing a polynucleotide, the method comprising contacting a template nucleic acid with a blend of DNA polymerases of  claim 20 .  
     
     
         41 . The method of  claim 38  wherein the engineered polymerase is engineered from at least Taq polymerase.  
     
     
         42 . The method of  claim 40  wherein an engineered polymerase in said blend is engineered from at least Taq polymerase.  
     
     
         43 . The method of  claim 38  wherein the engineered pol A polymerase is M1 or M4 as depicted in  FIG. 1   b  and  FIG. 2   b , respectively.  
     
     
         44 . The method of  claim 38  wherein said DNA polymerase is M1 DNA polymerase as depicted in  FIG. 1   b.    
     
     
         45 . The method of  claim 38  wherein said DNA polymerase is selected from the group consisting of the polymerases designated herein as: 3B5, 3B8, 3C12, 3D1, 3A10, 3B6, 3B11, 4D11 and 5D4.