In our phase I studies, we isolated a subassembly of a T. thermophilus DNA polymerase III holoenzyme, establishing the existence of a standard eubacterial replicase in extreme thermophiles. The objectives of our proposed phase II studies are to develop methods for obtaining large quantities of the Tth DNA polymerase III holoenzyme and, exploiting the enormous catalytic efficiency and processivity of this enzyme, to develop very long PCR techniques that enable routine amplification of 100 kb or longer sequences. Such an improvement in this important method will enhance basic molecular biology research and genome research by extending the size of the steps taken when walking along the genome. The availability of a suitable enzyme for very long PCR will enhance diagnostic procedures for cancer, pathogens and genetic defects and will greatly extend the technology for positional cloning and physical marker linkage analysis.
The development of a megabase processivity thermophilic replicase should have a dramatic impact on the 100. million dollar/year thermophilic polymerae market.
