This project proposes is that deficiencies in available DNA polymerases increase costs and limit the rate of DNA sequencing and that thermophilic phage DNA polymerases developed here will substantially improve automated fluorescent sequencing, as well as newer technologies being developed. PyroPhage(tm) DNA Polymerases greatly expand the molecular diversity and biochemical capabilities of available polymerases. Reliance on traditional microbiology approaches has prevented earlier discovery of these phage DNAPs. Hundreds of new PyroPhage DNAP genes have been discovered, ten of which have been expressed. The first of these has demonstrated several characteristics that should significantly improve sequencing sensitivity, reduce gaps, reduce errors and simplify template preparation. These including efficient synthesis through GC rich templates, efficient incorporation of at least two classes of chain terminators, incorporation of fluorescently labeled nucleotides, improved base calling of problematic dinucleotides, high efficiency of synthesis, efficient initiation at nicks, and the ability to use an RNA template. Based on a unique combination of biochemical attributes of the PyroPhage DNAP, we also propose the development of new approaches to Sanger sequencing to further improve throughput and sensitivity. We anticipate that Phase II will result in the development of one PyroPhage DNAP as the next generation enzyme for automate fluorescent sequencing, and three or more PyroPhage DNAPs as improved sequencing reagents for a variety of other platforms. The goal of this project is to reduce the cost and improve the reliability of methods for deciphering genomic sequences by providing a new class of enzymes that perform better in the sequencing reaction. It has been known for years that enzymes derived viruses that infect microbes (phage) have potential to reduce sequencing costs but none of the existing phage enzymes was stable enough to survive the high temperatures of the standard reaction and no means of isolating an appropriate enzyme existed. Using a new approach, this project has discovered hundreds of such enzymes and produced ten. One has been studied in detail and, as expected, it has characteristics that are expected to improve sequencing. ? ? ?
| Schoenfeld, Thomas W; Murugapiran, Senthil K; Dodsworth, Jeremy A et al. (2013) Lateral gene transfer of family A DNA polymerases between thermophilic viruses, aquificae, and apicomplexa. Mol Biol Evol 30:1653-64 |
| Moser, Michael J; DiFrancesco, Robert A; Gowda, Krishne et al. (2012) Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme. PLoS One 7:e38371 |
| Schoenfeld, Thomas; Liles, Mark; Wommack, K Eric et al. (2010) Functional viral metagenomics and the next generation of molecular tools. Trends Microbiol 18:20-9 |
