Rhett L. Affleck, James N. Demas, Peter M. Goodwin, James H. Jett,(1) Minq Wu and Richard A. Keller
Chemical Science and Technology Division, Los Alamos National Laboratory Los Alamos, NM 87545
The Los Alamos, flow cytometry based approach to DNA sequencing requires highly efficient single molecule detection. We have demonstrated a detection efficiency approaching 95% for single molecules delivered from a small source in the center of our flow cell. The efficiency for single molecule detection is limited by false positives from fluorescent impurities in buffers and enzyme solutions, radial diffusion of analyte molecules out of the center of the sample stream, and photobleaching of analyte molecules during their transit through the excitation laser beam.
We have also demonstrated that: (1) in-line photobleaching of fluorescent impurities present in the enzyme and buffer solutions reduces our background significantly and (2) a high molecular weight polymer added to the sheath stream forms complexes with the small analyte molecules in the sample stream resulting in reduced radial diffusion of the fluorescent adduct.
Details of these measurements and applications to our DNA sequencing project will be presented.
This work was supported by Los Alamos National Laboratory LDRD funds and the DOE/OHER Human Genome Program.
(1) Life Sciences Division