This project, as its R21 milestone, will deliver Taq DNA polymerases that catalyze the template-directed addition of nucleoside triphosphates carrying large fluorescent groups attached to their 3'-ends. The fluorescent groups therefore both terminate transiently the growth of the oligonucleotide chain, and signal the nature of the nucleotide that was last added. These polymerase variants will form the core of a """"""""cheap reagent"""""""" approach to the Sequencing by Synthesis (SbS) strategy. Gaining control over polymerase behavior is key for this approach to generate inexpensive genome-quality sequence data. The research will exploit a decade of experience in the Benner laboratory with nucleic acid analogs, polymerases that accept them, and practical application of the combination. The tactics assume that site directed and mutagenesis is generally site-directed damage, and therefore must be followed by directed evolution to obtain polymerase substrate combinations that meet specifications. Here, directed evolution will be used to restore catalytic power and fidelity in polymerases that have been engineered to accept fluorescent tags. We shall: (a) synthesize nucleoside triphosphates that have fluorescent blocking groups; (b) use directed evolution system in water-in-oil emulsions to select polymerases that accept the triphosphates efficiently and faithfully; (c) obtain polymerases to incorporate these to within 10%, the catalytic activity of native polymerases, and with specificity to better than one part in 10,000. Should this R21 milestones be passed, we will use the R33 year to develop a working prototype for a multiplexed sequencing-by-synthesis device using these polymerases.
The Aims will be to: (d) optimize the fluorescent compound-cleavage chemistry-polymerase combination, (e) use an artificially expanded genetic information system (AEGIS), the artificial alphabet invented in the Benner group, to bin primer-template combinations for parallel sequencing; and (f) exploit 2D gels to develop an architecture for a prototype parallel sequencing instrument based on the technologies developed in Aims 1-3 two dimensional arrays and in gels. ? ?
| Benner, Steven A (2012) Aesthetics in synthesis and synthetic biology. Curr Opin Chem Biol 16:581-5 |
| Havemann, Stephanie A; Hoshika, Shuichi; Hutter, Daniel et al. (2008) Incorporation of multiple sequential pseudothymidines by DNA polymerases and their impact on DNA duplex structure. Nucleosides Nucleotides Nucleic Acids 27:261-78 |
| Yang, Zunyi; Sismour, A Michael; Benner, Steven A (2007) Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages. Nucleic Acids Res 35:3118-27 |
| Yang, Zunyi; Sismour, A Michael; Sheng, Pinpin et al. (2007) Enzymatic incorporation of a third nucleobase pair. Nucleic Acids Res 35:4238-49 |
