Thermostable chimeric nucleic acid polymerases and uses thereof

Abstract

Novel thermostable chimeric nucleic acid polymerases and methods for their generation and use are disclosed. It is shown that these chimeric nucleic acid polymerases, such as DNA polymerases, can be constructed using enzymatically active domains, isolated from different proteins or chemically synthesized. It is demonstrated that chimeric nucleic acid polymerases of the present invention possess the chemical and physical properties of their component domains (e.g., exonuclease activity, thermostability) and that the chimeric polymerases are thermostable.

We claim: 
     
         1 . A method of synthesizing a recombinant nucleic acid encoding a thermostable chimeric nucleic acid polymerase having two, non-naturally associated, enzymatically active domains, the method comprising:
 (a) isolating a first nucleic acid encoding a first enzymatically active domain;   (b) isolating a second nucleic acid encoding a second enzymatically active domain, wherein said second enzymatically active domain is non-naturally associated with said first enzymatically active domain; and   (c) combining said first nucleic acid and said second nucleic acid to form said recombinant nucleic acid encoding said thermostable chimeric nucleic acid polymerase.   
     
     
         2 . The method of  claim 1 , wherein said first enzymatically active domain is a 3′-5′ exonuclease domain. 
     
     
         3 . The method of  claim 1 , wherein said second enzymatically active domain is a 5′-3′ polymerase domain. 
     
     
         4 . The method of  claim 1 , wherein said isolating in (a) and (b) comprises amplifying the first nucleic acid and the second nucleic acid by polymerase chain reaction (PCR) with a PCR primer comprising a first nucleotide sequence complementary to a terminal region of a 3′-5′ exonuclease domain of said first nucleic acid and a second nucleotide sequence complementary to a terminal region of a 5′-3′ polymerase domain of said second nucleic acid. 
     
     
         5 . The method of  claim 1 , wherein said combining comprises hybridizing said first nucleic acid to said second nucleic acid to form a composite polynucleotide template, and amplifying said composite polynucleotide template to form said recombinant nucleic acid encoding said thermostable chimeric nucleic acid polymerase. 
     
     
         6 . The method of  claim 2 , wherein said 3′-5′ exonuclease domain comprises a 3′-5′ exonuclease domain of Pho DNA polymerase. 
     
     
         7 . The method of  claim 6 , wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 396 of Pho DNA polymerase (SEQ ID NO:3). 
     
     
         8 . The method of  claim 2 , wherein said 3′-5′ exonuclease domain comprises a 3′-5′ exonuclease domain of Pwo DNA polymerase. 
     
     
         9 . The method of  claim 8 , wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 396 of Pwo DNA polymerase (SEQ ID NO:4). 
     
     
         10 . The method of  claim 8 , wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 421 of Pwo DNA polymerase (SEQ ID NO:5). 
     
     
         11 . The method of  claim 2 , wherein said 3′-5′ exonuclease domain comprises a 3′-5′ exonuclease domain of Sso DNA polymerase. 
     
     
         12 . The method of  claim 11 , wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 508 of Sso DNA polymerase (SEQ ID NO:6). 
     
     
         13 . The method of  claim 2 , wherein said 3′-5′ exonuclease domain comprises a 3′-5′ exonuclease domain of Tpac DNA polymerase. 
     
     
         14 . The method of  claim 13 , wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 395 of Tpac DNA polymerase (SEQ ID NO:16). 
     
     
         15 . The method of  claim 3 , wherein said 5′-3′ polymerase domain is a 5′-3′ polymerase domain of Taq DNA polymerase. 
     
     
         16 . The method of  claim 3 , wherein said 5′-3′ polymerase domain is a 5′-3′ polymerase domain of Tth DNA polymerase. 
     
     
         17 . The method of  claim 15 , wherein said 5′-3′ polymerase domain comprises amino acid residues 281 to 832 of Taq DNA polymerase (SEQ ID NO:1). 
     
     
         18 . The method of  claim 15 , wherein said 5′-3′ polymerase domain comprises amino acid residues 271 to 832 of Taq DNA polymerase (SEQ ID NO:7). 
     
     
         19 . The method of  claim 16 , wherein said 5′-3′ polymerase domain comprises amino acid residues 273 to 834 of Tth DNA polymerase (SEQ ID NO:2).