Compositions and methods utilizing DNA polymerases

Abstract

The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3′ to 5′ exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.

1 . An isolated recombinant family B DNA polymerase comprising a sequence selected from the sequences as indicated by accession numbers listed in Table II, and further comprising a mutation at a position corresponding to D141 and/or E143 of SEQ ID NO. 2.  
     
     
         2 . The isolated recombinant family B DNA polymerase of  claim 1 , wherein said said mutation at D141 is an aspartic acid (D) to threonine (T) or alanine (A) mutation, and said mutation at E143 is a glutamic acid (E) to alanine (A) mutation.  
     
     
         3 . The isolated recombinant family B DNA polymerase of  claim 1 , further comprising a mutation at a position corresponding to L408 and/or P410 of SEQ ID NO. 2.  
     
     
         4 . The isolated recombinant family B DNA polymerase of  claim 3 , wherein said mutation at L408 is a leucine (L) to histidine (H) or phenylalanine (F) mutation and said mutation at P410 is a proline (P) to leucine (P) mutation.  
     
     
         5 . The isolated recombinant family B DNA polymerase of  claim 1 , further comprising a mutation at one or more additional amino acids selected from the group of positions corresponding to A485, S345, T604, Y497, I630, E645, E578, R465, V401, N424, P569, E617, V640, S651, L396, E459, L456, E658, V437, L478, Y496, Y409 and A490 of SEQ ID NO: 2.  
     
     
         6 . The isolated recombinant family B DNA polymerase of  claim 5 , wherein said said mutation at S345 is serine (S) to proline (P), said mutation at A485 is alanine (A) to threonine (T), cysteine (C), serine (S), leucine (L), isoleucine (I), phenylalanine (F) or valine (V), said mutation at Y497 is tyrosine (Y) to cysteine (C), said mutation at I630 is isoleucine (I) to valine (V), said mutation at E645 is glutamic acid (E) to lysine (L), said mutation at E578 is glutamic acid (E) to lysine (L), said mutation at R465 is arginine (R) to methionine (M), said mutation at L396 is leucine (L) to glutamine (Q) or to proline (P), said mutation at S651 is serine (S) to asparagine (B), said mutation at L456 is leucine (L) to histidine (H), said mutation at Y496 is tyrosine (Y) to asparagine (B) or leucine (L), said mutation at Y409 is tyrosine (Y) to valine (V), said mutation at A490 is alanine (A) to tyrosine (Y).  
     
     
         7 . The isolated recombinant family B DNA polymerase of  claim 3 , further comprising a mutation at one or more additional amino acids selected from the group of positions corresponding to A485, S345, T604, Y497, I630, E645, E578, R465, V401, N424, P569, E617, V640, S651, L396, E459, L456, E658, V437, L478, Y496, Y409 and A490 of SEQ ID NO: 2.  
     
     
         8 . The isolated recombinant family B DNA polymerase of  claim 7 , wherein said said mutation at S345 is serine (S) to proline (P), said mutation at A485 is alanine (A) to threonine (T), cysteine (C), serine (S), leucine (L), isoleucine (I), phenylalanine (F) or valine (V), said mutation at Y497 is tyrosine (Y) to cysteine (C), said mutation at I630 is isoleucine (I) to valine (V), said mutation at E645 is glutamic acid (E) to lysine (L), said mutation at E578 is glutamic acid (E) to lysine (L), said mutation at R465 is arginine (R) to methionine (M), said mutation at L396 is leucine (L) to glutamine (Q) or to proline (P), said mutation at S651 is serine (S) to asparagine (B), said mutation at L456 is leucine (L) to histidine (H), said mutation at Y496 is tyrosine (Y) to asparagine (B) or leucine (L), said mutation at Y409 is tyrosine (Y) to valine (V), said mutation at A490 is alanine (A) to tyrosine (Y).  
     
     
         9 . The isolated recombinant family B DNA polymerase of  claim 1 , further comprising reduced discrimination against a non-conventional nucleotide selected from the group consisting of: dideoxynucleotides, ribonucleotides and conjugated nucleotides.  
     
     
         10 . The isolated recombinant family B DNA polymerase of  claim 9 , wherein said conjugated nucleotide is selected from the group consisting of radiolabeled nucleotides, fluorescently labeled nucleotides, biotin labeled nucleotides, chemiluminescently labeled nucleotides and quantum dot labeled nucleotides.  
     
     
         11 . The isolated recombinant family B DNA polymerase of  claim 3 , further comprising reduced discrimination against a non-conventional nucleotide selected from the group consisting of: dideoxynucleotides, ribonucleotides and conjugated nucleotides.  
     
     
         12 . The isolated recombinant family B DNA polymerase of  claim 11 , wherein said conjugated nucleotide is selected from the group consisting of radiolabeled nucleotides, fluorescently labeled nucleotides, biotin labeled nucleotides, chemiluminescently labeled nucleotides and quantum dot labeled nucleotides.  
     
     
         13 . An isolated recombinant family B DNA polymerase comprising at least two mutations selected from the group consisting of: an aspartic acid (D) to threonine (T) or alanine (A) mutation at position corresponding to D141 of SEQ ID NO. 2, a glutamic acid (E) to alanine (A) mutation at E143 of SEQ ID NO. 2, a proline (P) to leucine (L) mutation at P410 of SEQ ID NO. 2, a leucine (L) to Histidine (H) or phenylalanine (F) mutation at L408 of SEQ ID NO. 2, an alanine (A) to threonine (T) mutation at A485 of SEQ ID NO: 2, and a serine (S) to proline (P) mutation of SEQ ID NO. 2.  
     
     
         14 . A kit comprising an isolated recombinant family B DNA polymerase of  claim 1  and packaging material therefor.  
     
     
         15 . A kit comprising an isolated recombinant family B DNA polymerase of  claim 3  and packaging material therefor.  
     
     
         16 . A method of synthesizing a complementary strand of DNA, said method comprising: 
 contacting a template DNA molecule with a non-conventional nucleotide and a recombinant family B DNA polymerase of  claim 1;  and    incorporating said non-conventional nucleotide to synthesize a complementary DNA strand.    
     
     
         17 . A method of sequencing DNA comprising the steps of: 
 contacting a template DNA strand with a sequencing primer, a chain-terminating nucleotide analog, and a recombinant family B DNA polymerase of  claim 1;  and    incorporating said non-conventional nucleotide to synthesize a complementary DNA strand, wherein incorporation of said chain-terminating nucleotide analog results in the termination of chain elongation, such that the nucleotide sequence of said DNA strand is determined.    
     
     
         18 . The method of  claim 17 , wherein said chain-terminating nucleotide analog is a dideoxynucleotide.  
     
     
         19 . The method of  claim 18  wherein said dideoxynucleotide is detectably labeled.  
     
     
         20 . The method of  claim 19 , wherein said dideoxynucleotide is fluorescently labeled.  
     
     
         21 . The method of  claim 20 , wherein said dideoxynucleotide is labeled with a moiety selected from the group consisting of fluorescein and rhodamine.  
     
     
         22 . A method of synthesizing a complementary strand of DNA, said method comprising: 
 contacting a template DNA molecule with a non-conventional nucleotide and a recombinant family B DNA polymerase of  claim 3;  and    incorporating said non-conventional nucleotide to synthesize a complementary DNA strand.    
     
     
         23 . A method of sequencing DNA comprising the steps of: 
 contacting a template DNA strand with a sequencing primer, a chain-terminating nucleotide analog, and a recombinant family B DNA polymerase of  claim 3;  and    incorporating said non-conventional nucleotide to synthesize a complementary DNA strand, wherein incorporation of said chain-terminating nucleotide analog results in the termination of chain elongation, such that the nucleotide sequence of said DNA strand is determined.    
     
     
         24 . The method of  claim 23 , wherein said chain-terminating nucleotide analog is a dideoxynucleotide.  
     
     
         25 . The method of  claim 24  wherein said dideoxynucleotide is detectably labeled.  
     
     
         26 . The method of  claim 25 , wherein said dideoxynucleotide is fluorescently labeled.  
     
     
         27 . The method of  claim 26 , wherein said dideoxynucleotide is labeled with a moiety selected from the group consisting of fluorescein and rhodamine.