This Program Project is related to an effort at LI-COR begun in 1998 to develop a system for de novo sequencing of single DNA molecules with very long reads. In particular, the Program Project will further the development of reagents and microfluidics flowcells for the system. A successful system would be revolutionary with respect to speed, read length, cost and minimized laboratory infrastructure. An entire genome would be sequenced from a single genomic DNA sample without cloning or amplification, and the long reads not only enable de novo genome sequencing, but automatically provide haplotype information. We are targeting a per-instrument throughput of 500 raw base calls per second with low error rates (1 error per 10,000 finished bases with 5x coverage). Manufacturing cost for reagents and flowcells is initially targeted to be about 0.001 CENTS per FINISHED (5x) base, with the potential of laying the technological framework to enable future significant additional cost reductions. Concurrent instrumentation and image analysis developments are funded independently of this Program Project. The proposal has three Program Goals: 1) Design, fabricate, and evaluate multichannel flowcells that enable bead-docking of DNA templates; fluidic control of reagents (including polymerases and modified nucleotide substrates); and charge-switched partitioning of released labeled pyrophosphates from intact gamma-phosphate-labeled nucleotides. 2) Design, synthesize, and evaluate four modified nucleotide types (A,C,G,T) whereby such modification involves attaching a fluorescent dye with photophysics suitable for single molecule detection via various linker arm configurations to the gamma-phosphate of the nucleotide as well as the attachment of a charge moiety (e.g. +2 charge) to the nucleotide base. 3) Preparation, expression, purification and screening of mutant polymerase libraries to evolve a polymerase that is suitable for incorporating the charge-switch nucleotide substrates with a nucleotide incorporation rate and fidelity as appropriate for meeting the throughput goals in conjunction with the multichannel flowcells.
| Anderson, Jon P; Reynolds, Bambi L; Baum, Kristin et al. (2010) Fluorescent structural DNA nanoballs functionalized with phosphate-linked nucleotide triphosphates. Nano Lett 10:788-92 |
| Reynolds, Bambi; Miller, Rachel; Williams, John G et al. (2008) Synthesis and stability of novel terminal phosphate-labeled nucleotides. Nucleosides Nucleotides Nucleic Acids 27:18-30 |
| Williams, John G K; Steffens, David L; Anderson, Jon P et al. (2008) An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase-DNA complexes to surfaces. Nucleic Acids Res 36:e121 |
| Steffens, David L; Williams, John G K (2007) Efficient site-directed saturation mutagenesis using degenerate oligonucleotides. J Biomol Tech 18:147-9 |
