Disclosed and claimed are isolated
nucleic acid molecules encoding Bacillus stearothermophilus
DNA polymerase (DNApolI), including the
structural gene for DNApolI, such as DNApolI genes having insertions, deletions, inactivation, or mutations at the 5' end thereof and thus
encode Bst
polymerase I enzymes which lack or have reduced 3'-5'
exonuclease activity, as well as methods for making and using such
nucleic acid molecules and such polymerases. For instance, the
nucleic acid molecules are useful for making the polymerases, for example, by expression of a vector comprising the nucleic acid molecules; and, the polymerases are useful in
DNA sequencing and / or
labelling. Thus, disclosed and claimed
recombinant DNA clones corresponding to the Bacillus stearothermophilus
DNA polymerase (DNApolI)
structural gene with deletions at the 5' end. The polymerases from these
recombinant DNA clones exhibit
DNA synthesis domain activity but have reduced 3' to 5'
exonuclease activity. Further, minor modifications at the 5' and 3' ends allow the clones to be manipulated by
cloning and expressed as monomeric peptides. These deleted DNA clones give rise to truncated DNApolIK enzymes that are deficient in 3' to 5'
exonuclease activity and are useful in nucleic acid synthesis by
primer extension reactions, particularly DNA
labelling and
DNA sequencing reactions. Furthermore, these clones may be combined with other clones in
heterologous constructs to create
hybrid proteins.