DNA polymerase blends and mutant DNA polymerases

Abstract

A thermostable DNA polymerase composition comprising at least two DNA polymerases, one of which is substantially reduced in 5′-exonuclease activity and one of which has 5′-exonuclease activity. This polymerase may be used in methods including, but not limited to, nucleic acid synthesis, DNA sequencing, nucleic acid amplification and cDNA synthesis,

1 . An isolated recombinant nucleic acid molecule encoding a  Thermus filiformis  (Tfi) DNA polymerase having a D144A point mutation, wherein said point mutation substantially reduces the 5′-exonuclease activity of said polymerase.  
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , further comprising an E437D point mutation.  
     
     
         3 . An isolated mutant Tfi DNA polymerase produced from the nucleic acid molecule of  claim 1 .  
     
     
         4 . An isolated mutant Tfi DNA polymerase having a D144A point mutation.  
     
     
         5 . An isolated mutant Tfi DNA polymerase having D144A and E437D point mutations.  
     
     
         6 . A composition comprising at least two thermostable DNA polymerases wherein at least one of said polymerases is substantially reduced in 5′ exonuclease activity (exo−) and wherein at least one of said polymerases has 5′ exonuclease activity (exo+).  
     
     
         7 . The composition of  claim 6 , wherein said polymerases are from the same species of thermophilic bacteria.  
     
     
         8 . The composition of  claim 6 , wherein said 5′-exo− and said 5′-exo+ polymerases are combined in a ratio of between 9:1 and 1:9 (exo−:exo+).  
     
     
         9 . The composition of  claim 8 , wherein the ratio is 7:3 (exo−:exo+)  
     
     
         10 . A vector comprising the isolated nucleic acid molecule of  claim 1 .  
     
     
         11 . The vector of  claim 10  wherein said nucleic acid molecule is operably linked to a promoter.  
     
     
         12 . A host cell comprising the vector of  claim 11 .  
     
     
         13 . The composition of  claim 6 , further comprising at least two components selected from the group consisting of a detergent, buffer salt, deoxynucleoside triphosphate (dNTP) and dideoxynucleoside triphosphate (dNTP).  
     
     
         14 . The composition of  claim 13 , wherein said components are a detergent, buffer salt and dNTP.  
     
     
         15 . 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 recited in (a) in the presence of one or more deoxy- or didexoyribonucleoside triphosphates and the composition of  claim 6  under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule.    
     
     
         16 . A method of 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′-terminus of the first strand of said DNA molecule and said second primer is complementary to a sequence at or near the 3′-terminus 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 the composition of  claim 6 , under conditions such that the third strand complementary to said first strand and a fourth strand complementary to said second strand are synthesized;    (c) denaturing said first and third strands and said second and fourth strands; and    (d) repeating steps (a) to (c) one or more times.    
     
     
         17 . (canceled)  
     
     
         18 . (canceled)  
     
     
         19 . A method of preparing cDNA from mRNA, comprising: 
 (a) contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid; and    (b) contacting said hybrid formed in (a) with the composition of  claim 6  and dATP, dCTP, dGTP and dTTP, whereby a cDNA-RNA hybrid is obtained.    
     
     
         20 . A method of preparing dsDNA from mRNA, comprising: 
 (a) contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid; and    (b) contacting said hybrid formed in (a) with the composition of  claim 6 , dATP, dCTP, dGTP and dTTP, and an oligonucleotide or primer which is complementary to the first strand cDNA; whereby dsDNA is obtained.    
     
     
         21 . An isolated mutant Tfi DNA polymerase produced from the nucleic acid molecule of  claim 2.