Cloned DNA polymerases from thermotoga neapolitana

Abstract

The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga neapolitana (Tne). The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The present invention also relates to the cloning and expression of the Tne DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The Tne DNA polymerase of the invention may be used in well-known DNA sequencing and amplification reactions.

What is claimed is:  
     
         1 . A substantially pure  Thermotoga neapolitana  (Tne) DNA polymerase having a molecular weight of about 100 kilodaltons, or fragments thereof.  
     
     
         2 . The DNA polymerase of  claim 1 , which is isolated from  Thermotoga neapolitana.    
     
     
         3 . The DNA polymerase of  claim 2 , which is isolated from  Thermotoga neapolitana  DMS 5068.  
     
     
         4 . An isolated DNA molecule comprising a gene encoding a Tne DNA polymerase having a molecular weight of about 100 kilodaltons.  
     
     
         5 . An isolated DNA molecule of  claim 4 , wherein the gene is modified to reduce 3′-5′ exo activity.  
     
     
         6 . The isolated DNA molecule of  claim 4 , wherein the promoter of said gene is an inducible promoter.  
     
     
         7 . The isolated DNA molecule of  claim 6 , wherein said inducible promoter is selected from the group consisting of a λ-P L  promoter, a tac promoter, a trp promoter, and a trc promoter.  
     
     
         8 . A recombinant host comprising a gene encoding Tne DNA polymerase having a molecular weight of 100 kilodaltons.  
     
     
         9 . The recombinant host of  claim 8 , wherein said gene is obtained from  Thermotoga neapolitana.    
     
     
         10 . The recombinant host of  claim 9 , wherein said gene is obtained from  Thermotoga neapolitana  DMS 5068.  
     
     
         11 . The recombinant host of  claim 8 , wherein said host is prokaryotic.  
     
     
         12 . The recombinant host of  claim 11 , wherein said host is  E. coli.    
     
     
         13 . A method of producing a Tne DNA polymerase having a molecular weight of about 100 kilodaltons, said method comprising: 
 (a) culturing a cellular host comprising a gene encoding said DNA polymerase;    (b) expressing said gene; and    (c) isolating said DNA polymerase from said host.    
     
     
         14 . The method of  claim 13 , wherein said host is a eukaryotic host.  
     
     
         15 . The method of  claim 13 , wherein said host is a prokaryotic host.  
     
     
         16 . The method of  claim 15 , wherein said prokaryotic host is  E. coli.    
     
     
         17 . A method of synthesizing a double-stranded DNA molecule comprising: 
 (a) hybridizing a primer to a first DNA molecule; and    (b) incubating said DNA molecule of step (a) in the presence of one or more deoxyribonucleoside triphosphates and Tne DNA polymerase having a molecular weight of about 100 kilodaltons, under condition sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule.    
     
     
         18 . The method of  claim 17 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana.    
     
     
         19 . The method of  claim 18 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana.  DMS 5068.  
     
     
         20 . The method of  claim 17 , wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.  
     
     
         21 . The method of  claim 20 , wherein said host is a eukaryotic host.  
     
     
         22 . The method of  claim 20 , wherein said host is a prokaryotic host.  
     
     
         23 . The method of  claim 22 , wherein said prokaryotic host is  E. coli.    
     
     
         24 . The method of  claim 17 , wherein said deoxyribonucleoside triphosphates are selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, ddATP, ddCTP, ddGTP, ddlTP, ddTTP, [αS]dATP, [αS]dTTP, [αS]dGTP, and [αS]dCTP.  
     
     
         25 . The method of  claim 24 , wherein one or more of said deoxyribonucleoside triphosphates are detectably labeled.  
     
     
         26 . The method of  claim 25 , wherein said detectable label is selected from the group consisting of a radioactive isotope, a fluorescent label, a chemiluminescent label, a bioluminescent label, and an enzyme label.  
     
     
         27 . A method of sequencing a DNA molecule, comprising: 
 (a) hybridizing a primer to a first DNA molecule;    (b) contacting said DNA molecule of step (a) with deoxyribonucleoside triphosphates, Tne DNA polymerase having a molecular weight of about 100 kilodaltons, and a terminator nucleotide;    (c) incubating the mixture of step (b) under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule,    wherein said synthesized DNA molecules are shorter in length than said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 5′ termini; and (d) separating said synthesized DNA molecules by size so that at least a part of the nucleotide sequence of said first DNA molecule can be determined.    
     
     
         28 . The method of  claim 27 , wherein said terminator nucleotide is ddTTP.  
     
     
         29 . The method of  claim 27 , wherein said terminator nucleotide is ddATP.  
     
     
         30 . The method of  claim 27 , wherein said terminator nucleotide is ddGTP.  
     
     
         31 . The method of  claim 27 , wherein said terminator nucleotide is ddCTP.  
     
     
         32 . The method of  claim 27 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana.    
     
     
         33 . The method of  claim 32 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana  DMS 5068.  
     
     
         34 . The method of  claim 27 , wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.  
     
     
         35 . The method of  claim 27 , wherein one or more of said deoxyribonucleoside triphosphates is detectably labeled.  
     
     
         36 . The method of  claim 35 , wherein said labeled deoxyribonucleoside triphosphate is [α 35 S]dATP.  
     
     
         37 . A method for amplifying a double stranded DNA molecule, comprising: 
 (a) providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3′-termini of the first strand of said DNA molecule and said second primer is complementary to a sequence at or near the 3′-termini of the second strand of said DNA molecule;    (b) hybridizing said first primer to said first strand and said second primer to said second strand in the presence of Tne DNA polymerase having a molecular weight of about 100 kilodaltons, under conditions such that a third DNA molecule complementary to said first strand and a fourth DNA molecule complementary to said second strand are synthesized;    (c) denaturing said first and third strand, and said second and fourth strands with heat; and (d) repeating steps (a) to (c) one or more times.    
     
     
         38 . The method of  claim 37 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana.    
     
     
         39 . The method of  claim 38 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana  DMS 5068.  
     
     
         40 . The method of  claim 37 , wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA Polymerase.  
     
     
         41 . A kit for sequencing a DNA molecule, comprising: 
 (a) a first container means comprising a Tne DNA polymerase having a molecular weight of about 100 kilodaltons;    (b) a second container means comprising one or more dideoxyribonucleoside triphosphates; and    (c) a third container means comprising one or more deoxyribonucleoside triphosphates.    
     
     
         42 . The kit of  claim 41 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana.    
     
     
         43 . The kit of  claim 42 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana  DMS 5068.  
     
     
         44 . The kit of  claim 41 , wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.  
     
     
         45 . A kit for amplifying a DNA molecule, comprising: 
 (a) a first container means comprising a Tne DNA polymerase having a molecular weight of about 100 kilodaltons; and    (b) a second container means comprising one or more deoxyribonucleoside triphosphates.    
     
     
         46 . The kit of  claim 45 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana.    
     
     
         47 . The kit of  claim 46 , wherein said DNA polymerase is isolated from  Thermotoga neapolitana  DMS 5068.  
     
     
         48 . The kit of  claim 45 , wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.