Sequencing of the human genome created a large impact in medicine and biotechnology and opened up the realm of personal therapeutics. A major progress-limiting factor is the inherent cost of current technologies reaching millions of dollars per mammalian genome. Our approach is based on a novel implementation of the proven Sanger method for DNA sequencing. We rely on nanotechnology to greatly increase throughput, reduce reagent volumes and drive costs down by orders of magnitude. In preliminary work, we have demonstrated the feasibility of a new approach for electrophoretic separation of DNA fragments along the surface of an Atomic Force Microscope probe tip. We showed that based on this surface electrophoresis method strands up to at least 100 bases can be separated in size with sample quantities down to only a few molecules and with speeds that are four to five orders of magnitude faster than in conventional capillary electrophoresis . We now propose to evolve this methodology toward a complete DNA sequencing scheme based on the Sanger method. Fluorescently labeled DNA fragments resulting from the Sanger reactions are sorted by size into bands . We detect their passage at the end of the probe tip using a novel optical detection scheme. We envision a massively parallel mode of operation in an array of 100x100 probe tips.
We aim to develop technology that can ultimately reach the goal of $1000 per genome by the parallel operation of probe tip sequencers with high analysis throughput and ultimately very small volumes of reagents.
The specific aims of this research are the following:
Aim 1. We demonstrate a scheme for DNA sequencing based on single probe tip electrophoretic separation of DNA fragments up to 100bp.
Aim 2. To demonstrate scalability, we address the parallel operation of DNA sequencers with a linear array of 10 probe tips.
| Nawarathna, D; Chang, R; Nelson, E et al. (2011) Targeted messenger RNA profiling of transfected breast cancer gene in a living cell. Anal Biochem 408:342-4 |
