|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
23. Analysis of Gradients of Polymer Concentration or Ionic Strength
Mark A. Quesada, David J. Fisk,
and F. William Studier
We are investigating whether gradients of polymer concentration or ionic strength can extend read lengths into the 1000-2000 base range when analyzing DNA sequencing reactions by capillary electrophoresis. Longer read lengths would increase sequencing efficiency and reduce the effort needed in the assembly and finishing stages of genome sequencing.
Gradients of polymer concentration or ionic strength along the length of the capillary are generated by merging two solutions in the capillary, using programmable syringe pumps. Parameters important for obtaining reproducible gradients were identified and controlled with the aid of fluorescent dye to analyze the distribution of one of the solutions along the length of the capillary. Because entangled polymer solutions have high viscosity, balancing hydrostatic conductance at the junction between the merging solutions is critical for producing well defined, reproducible gradients. A smooth gradient with uniform composition in the radial direction at each capillary cross section is established by radial diffusion, primarily of water and other low molecular weight components within the capillary (polymer swelling). Production of reproducible gradients requires merging the solutions in a controlled way at a sufficiently low flow rate, and allowing sufficient time for diffusion to create a smooth and uniform gradient before the capillary is used for analysis.
Once conditions were established for preparing reproducible and useful gradients of polymer concentration in capillaries, the effects of different gradient configurations on read length were examined. We expected that a gradient of increasing polymer concentration could counter the peak-broadening effects that are responsible for loss of resolution at long read lengths, and this appears to be the case. Certain gradient configurations yield single-base resolution near 800 bases in standard, room-temperature analyses with separations continuing well beyond 1000 bases. Gradients of increasing ionic strength (salt concentration) might be expected to have a similar band sharpening effect, and we are beginning to explore resolution in different combinations of polymer and salt gradients.
If the band sharpening effects of polymer or salt gradients can increase resolution and extend DNA read lengths in capillary electrophoresis, the same concepts may also find application for improving the performance of microchannel systems and disposable chips.
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