Bacillus stearothermophilus DNA polymerase I (klenow) clones including those with reduced 3'-to-5' exonuclease activity

Abstract

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 expressior 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.