R.L. Affleck, J.N. Demas, P.M. Goodwin, J.H. Jett, R.A. Keller, J.C. Martin, J.A. Schecker, D.J. Semin, and M. Wu
Center for Human Genome Studies, Los Alamos National Laboratory
We are developing a technique to determine the sequence of bases in large fragments of DNA. Our goal is a sequencing rate 100 to 1000 bases per second on DNA strands approaching 40 kb in length. The ideas presented represent the combined effort of a multidisciplinary team composed of physicists, physical chemists, cellular and molecular biologists and organic chemists. A large fragment of DNA, approximately 40 kb in length, will be labeled with base identifying fluorescent tags and suspended in the flow stream of a flow cytometer capable of single fluorescent molecule detection. The tagged bases will be cleaved sequentially from the single DNA fragment and identified by laser-induced fluorescence as they pass through the excitation laser beam. We have demonstrated that each of the component parts of the technology work separately and have integrated them into a prototype instrument. Using this prototype, we have demonstrated recently the digestion of fluorescently labeled DNA fragments anchored in a flowing stream containing E. coli exonuclease III. We have detected single labeled nucleotides, enzymatically cleaved from 15-30 DNA fragments anchored in the flow stream. This is a major milestone towards the realization of DNA sequencing in flow. From this data we estimate the exonuclease turnover rate in this system to be approximately 5 nucleotides per second per DNA strand at 36°C, similar to the rate measured under static conditions for this exonuclease and native DNA. We are exploring different ways to increase the exonuclease turnover rate to approximately 100 nucleotides per second per DNA strand.