Richard A. Mathies, Steve M. Clark, Andrew J. de Mello, Brian B. Haab, Jingyue Ju, Indu Kheterpal, James R. Scherer, Yiwen Wang and Adam T. Woolley
Department of Chemistry, University of California, Berkeley CA 94720
This presentation will focus on the portion of our work concerned with the development of miniaturized capillary array electrophoresis (CAE) chips and integrated DNA analysis devices as well as the practical extension of detection sensitivity to the single molecule limit.
CAE Chips. To reduce the electrophoretic lane dimensions and to increase separation speed we have developed microfabricated capillary arrays using photolithographic masking and chemical etching techniques. High resolution restriction and PCR fragment sizing separations can be performed on these chips in under 120 s. Using polymerized gels, these chips can separate DNA sequencing reactions out to 400 bases in under 10 minutes. Raw sequencing rates for a single microfabricated capillary are ~1000-2000 bases/hour. We are currently working with a 32 capillary chip that can perform simultaneous separations of 32 different samples in parallel. We are also working on integrating PCR sample preparation on these chips to produce miniaturized integrated DNA analysis systems.
Single Molecule DNA Fragment Detection. The goal of this research is to enhance the sensitivity of trace DNA fragment detection. Toward this end we have devised and recently demonstrated the detection of dsDNA fragments by using single-molecule fluorescence burst counting. A confocal detection system was used to observe fluorescence bursts from single molecules of dsDNA multiply labeled with the intercalation dye TO6. Flowing solutions of M13 DNA were first used to show that the number of bursts was linear with concentration, that the average burst duration was consistent with the expected transit time, and that the number of detected bursts was consistent with the concentration. The optimized single molecule apparatus and analysis method was then used to detect CE separations of M13, pBR 322 and pRL 277 DNA. Separations are easily detected when only 50100 molecules of DNA per band pass through the detection region, and the current detection size limit is ò100 bp/fragment. This new detection technology should lead to the routine analysis of DNA with an on-column sensitivity better than 100 molecules/band. Applications to cancer, bacterial, viral and trace expression detection are envisioned.
The U. C. Berkeley High Sensitivity DNA Analysis Project (directed by R. A. Mathies and A. N. Glazer) was supported by the U. S. Department of Energy under contract DE-FG-91ER61125.
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2. Zhu, H., Clark, S. M., Benson, S. C., Rye, H. S., Glazer, A. N. and Mathies, R. A. High-Sensitivity Capillary Electrophoresis of Double-Stranded DNA Fragments using Monomeric and Dimeric Fluorescent Intercalation Dyes, Analytical Chemistry 66, 1941-1948 (1994).
3. Woolley, A. T. and Mathies, R. A. Ultra-High-Speed DNA Fragment Separations Using Microfabricated Capillary Array Electrophoresis Chips, Proc. Natl. Acad. Sci. U.S.A. 91, 11348-11352 (1994).
4. Wang, Y., Ju, J., Carpenter, B., Atherton, J. M., Sensabaugh, G. F. and Mathies, R. A. High-Speed, High-Throughput THO1 Allelic Sizing Using Energy Transfer Fluorescent Primers and Capillary Array Electrophoresis, Analytical Chemistry 67, 1197-1203 (1995).
5. Ju, J., Ruan, C., Fuller, C. W. Glazer, A. N. and Mathies, R. A. Fluorescence Energy Transfer Dye-Labeled Primers for DNA Sequencing and Analysis, Proc. Natl. Acad. Sci. U.S A. 92, 4347-4351 (1995).
6. Woolley, A. T. and Mathies, R. A. Ultra-High-Speed DNA Sequencing Using Capillary Array Electrophoresis Chips, Proceedings of the International Society for Optical Engineering-SPIE, Volume 2386, 36-44 (1995).
7. Ju, J., Kheterpal, I., Scherer, J. R., Ruan, C., Fuller, C. W., Glazer, A. N. and Mathies, R. A. Design and Synthesis of Fluorescence Energy Transfer Dye-Labeled Primers and their Application for DNA Sequencing and Analysis, Analytical Biochemistry 231, 131-140 (1995).
8. Woolley, A. T. and Mathies, R. A. Ultra-High-Speed DNA Sequencing Using Capillary Electrophoresis Chips, Analytical Chemistry 67, 3676-3680 (1995).
9. Haab, B. B. and Mathies, R. A. Single Molecule Fluorescence Burst Detection of DNA Fragments Separated by Capillary Electrophoresis, Analytical Chemistry 67, 3253-3260 (1995).