DNA polymerases are the engines that drive conventional methods of DNA sequence analysis, as well as many of the newer sequencing methods being developed. Variants of Taq DNA polymerase have been used for nearly all genomic sequencing to date. However, Taq DNA polymerase has a number of inherent biochemical deficiencies that limit the rate and reliability of sequence analysis and increase its cost. In this project, a highly processive Taq polymerase will be created by fusing a DNA binding domain to the N-terminus of the enzyme. This modified polymerase, with optimized protocols, will improve the ability to sequence from trace amounts of DNA, enabling direct sequence analysis from single bacterial colonies (""""""""ex cyto"""""""" sequencing). Analogous to colony PCR, ex cyto sequencing will eliminate the need for overnight growth of bacterial cultures, expensive template purification, and purchase of purified DNA polymerase, dramatically decreasing the cost of DNA sequence analysis. A Taq DNA polymerase with high processivity will also improve technical aspects of the sequencing process, such as reading through """"""""difficult"""""""" templates and providing increased read lengths, thereby improving the accuracy of called bases and their subsequent assembly. Important advances in medical science and basic research will result from faster and cheaper access to higher quality genomic sequence data of numerous organisms. Project Narrative: The success of the human genome project has spawned explosive growth in the demand for DNA sequence information. The discovery of new genes from a variety of species will have a large impact on understanding human health and disease. Despite improvements in speed and reduction in costs of DNA sequence analysis, the process is still time, labor, and cost-intensive. This proposal seeks to dramatically improve the speed while decreasing the costs of DNA sequencing. ? ? ?
