|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
39. Integrated Sequencing Sample Preparation on CE Microplates
Yining Shi1, Indu Kheterpal1,
Jin Xie2, Alexander N. Glazer2, and Richard A. Mathies1
Microfabricated devices are revolutionizing the field of DNA electrophoresis because DNA fragments can now be separated in less than 1 minute1,2 and sequencing separations are achieved in ~10 minutes3. Furthermore, Microfabricated devices allow the integration of sample preparation, clean-up, separation and detection. To achieve this goal we have performed high quality sequencing separations on microchannels and are developing solid-phase microfluidic methods to concentrate and clean up DNA samples for efficient injection into the separation columns.
The quality of separation of DNA fragments is highly dependent on the injection and separation conditions. We have optimized four-color sequencing on microfabricated capillary electrophoretic devices for separation matrix, temperature, channel dimensions, injector size and injection parameters. Linear polyacrylamide (LPA; 4%) matrices were used to achieve sequencing separations of 600 bases on 7 cm long channels in ~20 minutes. The sequence data were analyzed and base-called using BaseFinder4 and an accuracy rate of 99.4% was obtained to 500 bases3.
We are now developing methods to integrate these excellent separations with sample preparation methods on a single device. We have synthesized biotinylated energy transfer primers for fragment amplification and sequencing5. The presence of the biotin allows us to utilize solid-phase surface chemistry to purify and concentrate DNA samples before introducing them into the separation columns. We have successfully constructed sandwich structures of biotin-streptavidin-biotin on the glass surface. The biotinylated PCR products are pumped through a capture chamber and concentrated onto the surface containing biotin-streptavidin. The products captured in the reaction chamber are cleaned, denatured with formamide at 90C and injected directly into the separation columns. These sample clean-up methods are relevant to developing fully integrated microdevices.
1 Woolley, A. T. and Mathies, R. A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11348-11352.
2 Simpson, P. C.; Roach, D.; Woolley, A. T.; Thorsen, T.; Johnston, R.; Sensabaugh, G. F. and Mathies, R. A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2256-2261.
3 Liu, S.; Shi, Y.; Ja, W.; Mathies, R. A. (1998) Anal. Chem. in press.
4 Giddings, M. C.; Severin, J; Westphall, M; Wu, J. Z; and Smith, L. M. (1998) Genome Research 8, 644-665.
5 See abstract "Synthesis, Characterization, and Potential Applications of Biotinylated Energy Transfer Oligonucleotides" by J. Xie, R. A. Mathies, A. N. Glazer.
|Author Index||Sequencing Technologies & Resources||Microbial Genome Program|
|Search||Mapping||Ethical, Legal, & Social Issues|