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 . A composition for identifying a nucleotide at a given position of a template DNA molecule, said composition comprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides and a first primer, wherein said first primer anneals to the immediate 3′ of said nucleotide at the given position of said template DNA molecule.  
     
     
         2 . The composition of  claim 1 , wherein said Family B DNA polymerase is a JDF-3 DNA polymerase.  
     
     
         3 . The composition of  claim 2 , wherein said JDF-3 DNA polymerase has a sequence of SEQ ID NO: 2 and further comprises one or more amino acid mutations at D141, E143, A485, L408 or P410.  
     
     
         4 . The composition of  claim 3 , wherein said JDF-3 DNA polymerase has one or more amino acid mutations selected from the group consisting of: D141A or D141T, E143A, L408H or L408F, A485T, and P410L.  
     
     
         5 . The composition of  claim 3 , wherein said JDF-3 DNA polymerase is substituted at position P410 with an amino acid having a non polar side chain.  
     
     
         6 . The composition of  claim 5 , wherein said amino acid having a non polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.  
     
     
         7 . The composition of  claim 4 , wherein said JDF-3 DNA polymerase comprises four amino acid mutations at D141A, E 143A, P410L and A485T.  
     
     
         8 . The composition of claim  1 - 6 , wherein said Family B DNA polymerase is deficient in 3′ to 5′ exonuclease activity.  
     
     
         9 . The composition of  claim 1 , further comprising at least one chain-terminating nucleotide analog, wherein said chain-terminating nucleotide analog is incorporated into said first primer by said Family B DNA polymerase in a template-dependent manner.  
     
     
         10 . The composition of  claim 1 , wherein at least one chain-terminating nucleotide analog is labeled with a first detectable label.  
     
     
         11 . The composition of  claim 1 , wherein more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.  
     
     
         12 . The composition of  claim 9 , wherein said chain-terminating nucleotide analog is a dideoxynucleotide.  
     
     
         13 . The composition of  claim 12 , wherein said dideoxynucleotide is selected from the group consisting of: ddATP, ddTTP, ddCTP and ddGTP.  
     
     
         14 . The composition of  claim 9 , wherein said first primer is labeled with a second detectable label.  
     
     
         15 . The composition of  claim 14 , wherein first and second detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.  
     
     
         16 . The composition of  claim 1 , further comprising a second primer.  
     
     
         17 . The composition of  claim 16 , wherein said first primer is labeled with a second detectable label and said second primer is labeled with a third detectable label, said second and third detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.  
     
     
         18 . The composition of  claim 17 , wherein said second primer anneals to the immediate 5′ of said nucleotide at the given position of said template DNA molecule.  
     
     
         19 . The composition of  claim 18 , further comprising a DNA ligase.  
     
     
         20 . The composition of  claim 1 , further comprising a reaction buffer for said Family B DNA polymerase.  
     
     
         21 . The composition of  claim 1 , wherein said template DNA molecule is the product of a polymerase chain reaction or a plasmid DNA.  
     
     
         22 . The composition of any one of claims  10 ,  14 , or  17 , wherein said first or second or third detectable label is one selected from the group consisting of: a fluorescent label, an isotope, a chemiluminescent label, a quantum dot label, an antigen, or an affinity moiety.  
     
     
         23 . The composition of  claim 22 , wherein said first detectable label is a rhodamine label or a cyanine label.  
     
     
         24 . An isolated recombinant Family B DNA polymerase having reduced discrimination against non-conventional nucleotides, wherein said DNA polymerase comprises an amino acid mutation at a position corresponding to P410 of SEQ ID NO:2.  
     
     
         25 . The isolated recombinant Family B DNA polymerase of  claim 24 , wherein said amino acid mutation substitution with an amino acid having a non-polar side chain.  
     
     
         26 . The isolated recombinant Family B DNA polymerase of  claim 25 , wherein said amino acid having a non-polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.  
     
     
         27 . An isolated recombinant Family B DNA polymerase having reduced discrimination against non-conventional nucleotides, wherein said DNA polymerase comprises amino acid mutations in the P Helix and at a position corresponding to P410 of SEQ ID NO: 2.  
     
     
         28 . The isolated recombinant Family B DNA polymerase of  claim 27 , wherein said amino acid mutation in the P Helix is located at a position corresponding to A485 of SEQ ID NO: 2.  
     
     
         29 . The isolated recombinant Family B DNA polymerase of  claim 28 , wherein said amino acid mutation at a position corresponding to P410 of SEQ ID NO: 2 is a substitution with an amino acid having a non-polar side chain.  
     
     
         30 . The isolated recombinant Family B DNA polymerase of  claim 29 , wherein said amino acid having a non-polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.  
     
     
         31 . A kit for identifying a nucleotide at a given position of a template DNA molecule, said kit comprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides and a first primer, wherein said first primer anneals to the immediate 3′ of said nucleotide at the given position of said template DNA molecule.  
     
     
         32 . The kit of  claim 31 , wherein said Family B DNA polymerase is a JDF-3 DNA polymerase.  
     
     
         33 . The kit of  claim 32 , wherein said JDF-3 DNA polymerase has a sequence of SEQ ID NO: 2 and further comprises one or more amino acid mutations at D141, E143, A485, L408 or P410.  
     
     
         34 . The kit of  claim 32 , wherein said JDF-3 DNA polymerase is substituted at a position corresponding to P410 of SEQ ID No: 2 with an amino acid having a non-polar side chain.  
     
     
         35 . The kit of  claim 34 , wherein said amino acid having a non-polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.  
     
     
         36 . The kit of  claim 33 , wherein said JDF-3 DNA polymerase has one or more amino acid mutations selected from the group consisting of: D141A or D141T, E143A, L408H or L408F, A485T, and P410L.  
     
     
         37 . The kit of  claim 36 , wherein said JDF-3 DNA polymerase comprises four amino acid mutations at D141A, E 143A, P410L and A485T.  
     
     
         38 . The kit of  claim 31 , wherein said Family B DNA polymerase is deficient in 3′ to 5′ exonuclease activity.  
     
     
         39 . The kit of  claim 31 , further comprising at least one chain-terminating nucleotide analog, wherein said chain-terminating nucleotide analog is incorporated into said first primer by said Family B DNA polymerase in a template-dependent manner.  
     
     
         40 . The kit of  claim 39 , wherein at least one chain-terminating nucleotide analog is labeled with a first detectable label.  
     
     
         41 . The kit of  claim 39 , wherein more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.  
     
     
         42 . The kit of  claim 39 , wherein said chain-terminating nucleotide analog is a dideoxynucleotide.  
     
     
         43 . The kit of  claim 42 , wherein said dideoxynucleotide is selected from the group consisting of: ddATP, ddTTP, ddCTP and ddGTP.  
     
     
         44 . The kit of  claim 39 , wherein said first primer is labeled with a second detectable label.  
     
     
         45 . The kit of  claim 44 , wherein first and second detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.  
     
     
         46 . The kit of  claim 31 , further comprising a second primer.  
     
     
         47 . The kit of  claim 46 , wherein said first primer is labeled with a second detectable label and said second primer is labeled with a third detectable label, said second and third detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.  
     
     
         48 . The kit of  claim 47 , wherein said second primer anneals to the immediate 5′ of said nucleotide at the given position of said template DNA molecule.  
     
     
         49 . The kit of  claim 48 , further comprising a DNA ligase.  
     
     
         50 . The kit of  claim 31 , further comprising a reaction buffer for said Family B DNA polymerase.  
     
     
         51 . The kit of  claim 31 , wherein said template DNA molecule is the product of a polymerase chain reaction or a plasmid DNA.  
     
     
         52 . The kit of  claim 40 ,  44 , or  47 , wherein said first or second or third detectable label is one selected from the group consisting of: a fluorescent label, an isotope, a chemiluminescent label, a quantum dot label, an antigen, or an affinity moiety.  
     
     
         53 . The kit of  claim 52 , wherein said first detectable label is a rhodamine label or a cyanine label.  
     
     
         54 . The kit of  claim 31 , further comprising a control template and/or at least one control primer.  
     
     
         55 . The kit of  claim 54 , comprising a control template and four control primers.  
     
     
         56 . A kit for identifying a nucleotide at a given position of a template DNA molecule, said kit comprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides, wherein said DNA polymerase comprises an amino acid mutation at a position corresponding to P410 of SEQ ID NO:2.  
     
     
         57 . The kit of  claim 56 , wherein said amino acid mutation at a position corresponding to P410 of SEQ ID NO:2 is an amino acid substitution with an amino acid having a non polar side chain.  
     
     
         58 . The kit of  claim 57 , wherein said amino acid having a non polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.  
     
     
         59 . A kit for identifying a nucleotide at a given position of a template DNA molecule, said kit comprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides, wherein said DNA polymerase comprises an amino acid mutation in the P Helix and at a position corresponding to P410 of SEQ ID NO: 2.  
     
     
         60 . The kit of  claim 59 , wherein said amino acid mutation in the P Helix is located at a position corresponding to A485 of SEQ ID NO: 2.  
     
     
         61 . The kit of  claim 59  wherein the mutation at a position corresponding to P410 of SEQ ID NO: 2 is a substitution with an amino acid with a non-polar side chain.  
     
     
         62 . The kit of  claim 61 , wherein said amino acid having a non polar side chain is selected from the group of amino acid selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.  
     
     
         63 . A method of identifying a nucleotide at a given position of a template DNA molecule in a sample, said method comprising: 
 (a) contacting a first primer with said template DNA molecule, wherein said contacting allows said first primer to anneal to the immediate 3′ of said nucleotide at the given position of said template DNA molecule, so as to form a duplex between said first primer and said template DNA molecule;    (b) incubating said duplex from step (a), in the presence of a Family B DNA polymerase and at least one chain-terminating nucleotide analog, said Family B DNA polymerase having reduced discrimination against non-conventional nucleotides and said terminator is labeled with a first detectable label, wherein said incubating allows the incorporation of a labeled chain-terminating nucleotide analog into said first primer by said DNA polymerase in a template-dependent manner; and    (c) determining the presence or identity of said duplex from step (b) by a signal generated from said first detectable label.    
     
     
         64 . The method of  claim 63 , wherein said Family B DNA polymerase is a JDF-3 DNA polymerase.  
     
     
         65 . The method of  claim 64 , wherein said JDF-3 DNA polymerase has a sequence of SEQ ID NO: 2 and further comprises one or more amino acid mutations at D141, E143, A485, L408 or P410.  
     
     
         66 . The method of  claim 64 , wherein said JDF-3 DNA polymerase is substituted at position P410 with an amino acid having a non polar side chain.  
     
     
         67 . The method of  claim 66 , wherein said amino acid having a non polar side chain is methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.  
     
     
         68 . The method of  claim 64 , wherein said JDF-3 DNA polymerase has one or more amino acid mutations selected from the group consisting of: D141A or D141T, E143A, L408H or L408F, A485T, and P410L.  
     
     
         69 . The method of  claim 68 , wherein said JDF-3 DNA polymerase comprises four amino acid mutations at D141A, E 143A, P410L and A485T.  
     
     
         70 . The method of  claim 63 , wherein said Family B DNA polymerase is deficient in 3′ to 5′ exonuclease activity.  
     
     
         71 . The method of  claim 63 , wherein at least one chain-terminating nucleotide analog is labeled with a first detectable label.  
     
     
         72 . The method of  claim 63 , wherein more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.  
     
     
         73 . The method of  claim 63 , wherein said chain-terminating nucleotide analog is a dideoxynucleotide.  
     
     
         74 . The method of  claim 73 , wherein said dideoxynucleotide is selected from the group consisting of: ddATP, ddTTP, ddCTP and ddGTP.  
     
     
         75 . The method of  claim 63 , wherein said first primer is labeled with a second detectable label.  
     
     
         76 . The method of  claim 75 , wherein first and second detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.  
     
     
         77 . The method of  claim 63 , wherein said template DNA molecule is the product of a polymerase chain reaction or a plasmid.  
     
     
         78 . The method of  claim 77 , further comprising removing PCR primers and dNTPs from the PCR product before step (a).  
     
     
         79 . A method of identifying a nucleotide at a given position of a template DNA molecule in a sample, said method comprising: 
 a) contacting a first primer and s second primer with said template DNA molecule, wherein said contacting allows said first primer to anneal to the immediate 3′ of said nucleotide at the given position of said template DNA molecule and said second primer to anneal to the immediate 5′ of said nucleotide at the given position of said template DNA molecule, so as to form a complex between said template DNA molecule and said first and second primers, said first primer being labeled with a second detectable label and said second primer being labeled with a third detectable label.    b) incubating said complex from step (a), in the presence of a DNA ligase, wherein said incubating allows the ligation between said first and second primers so as to form a single molecule; and    c) determining the presence or identity of said single molecule from step (b) by a signal generated from said second and third detectable labels.    
     
     
         80 . The method of  claim 63 ,  75 , or  79 , wherein said first or second or third detectable label is one selected from the group consisting of: a radiolabel, a fluorescent label, a chemiluminescent label, a colorimetric label and an enzymatic label.  
     
     
         81 . The method of  claim 80 , wherein said first detectable label is a rhodamine label or a cyanine label.  
     
     
         82 . A method of identifying a nucleotide at a given position of a template DNA molecule in a sample, said method comprising: 
 a) contacting a first primer with said template DNA molecule, wherein said contacting allows said first primer to anneal to the immediate 3′ of said nucleotide at the given position of said template DNA molecule, so as to form a duplex between said first primer and said template DNA molecule;    b) incubating said duplex from step (a), in the presence of a Family B DNA polymerase and at least one chain-terminating nucleotide analog, said Family B DNA polymerase having reduced discrimination against non-conventional nucleotides, wherein said DNA polymerase comprises an amino acid mutation in a position corresponding to P410 of SEQ ID NO: 2 and said terminator is labeled with a first detectable label, wherein said incubating allows the incorporation of a labeled chain-terminating nucleotide analog into said first primer by said DNA polymerase in a template-dependent manner; and    c) determining the presence or identity of said duplex from step (b) by a signal generated from said first detectable label.    
     
     
         83 . The method of  claim 82 , wherein said amino acid mutation at a position corresponding to P410 of SEQ ID NO:2 is an amino acid substitution with an amino acid having a non polar side chain.  
     
     
         84 . The method of  claim 83 , wherein said amino acid having a non polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.  
     
     
         85 . A method of identifying a nucleotide at a given position of a template DNA molecule in a sample, said method comprising: 
 (a) contacting a first primer with said template DNA molecule, wherein said contacting allows said first primer to anneal to the immediate 3′ of said nucleotide at the given position of said template DNA molecule, so as to form a duplex between said first primer and said template DNA molecule;    (b) incubating said duplex from step (a), in the presence of a Family B DNA polymerase and at least one chain-terminating nucleotide analog, said Family B DNA polymerase having reduced discrimination against non-conventional nucleotides, wherein said DNA polymerase comprises amino acid mutations in the P Helix and at a position corresponding to P410 of SEQ ID NO:2 and said terminator is labeled with a first detectable label, wherein said incubating allows the incorporation of a labeled chain-terminating nucleotide analog into said first primer by said DNA polymerase in a template-dependent manner; and 
 determining the presence or identity of said duplex from step (b) by a signal generated from said first detectable label.  
   
     
     
         86 . The method of  claim 85 , wherein said amino acid mutation in the P Helix is located at a position corresponding to position A485 of SEQ ID NO: 2.  
     
     
         87 . The method of  claim 86 , wherein said amino acid mutation at a position corresponding to position P410 of SEQ ID NO: 2 is an amino acid substitution with an amino acid having a non polar side chain.  
     
     
         88 . The method of  claim 87 , wherein said amino acid having a non polar side chain is selected from the group of methionine, glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.