Brian Paegel
         

 

Ph.D. Candidate, UC Berkeley Chemistry Dept.

B. S. Chemistry, Duke University, 1998

Personal Web Page: http://www.cchem.berkeley.edu/~ramgrp/brian/
 
           

DNA Migration Dynamics in Microchannels with Complex Geometries

Microfabricated capillary electrophoresis systems are the next generation in high-throughput, nanovolume sample handling and separation. We have demonstrated fundamental applications in DNA analysis on electrophoresis microchips as well as novel microfabricated capillary arrays for massively parallel separations. As the feature density and separation performance requirements of these devices increases, it becomes necessary to fold the separation channel back on itself in a serpentine design. The turns used in these designs degrade separation performance, which has provided the impetus to investigate turn structures that maintain the efficiency of the separation. Theory has shown that the main factor responsible for decrease in separation efficiency in folded channels is the width of the channel in the turn. We have constructed a device with folded channels where the turn portion of the channel has a constricted width. The channel is tapered down to the constricted width just prior to the turn, and then widened to the original width at the turn exit. A test device in which we used our radial confocal fluorescence scanner to monitor the same separation simultaneously at three points along a folded channel has shown us the optimal parameters governing the performance of these tapered turns. The image data displayed below were collected on the scanning microscope system and demonstrates the improved efficacy of a channel utilizing "hyper-turns" over a U-shaped turn in separating the fragments of a standard DNA sizing ladder.
           
Left: A serpentine channel containing two U-shaped turns is compared to the same length channel containing two hyper-turns. In both cases, the DNA sample is injected from the intersection at top and move through the serpentine in the direction of the arrow. Upon traversing the first turn, the bands are tilted in the U-shaped turn, as predicted by theory. This tilting is due to the "racetrack effect," a reference to the observation that a car, for example, traversing the inside radius of a turn will complete the turn faster than a car on the outer radius of the turn. Since the distance difference gradiates linearly over the width of the channel, bands are tilted linearly in the transverse dimension. The hyper-turns, with constricted channel widths in the turn, essentially force all molecules in the sample plug to follow the same radius through the turn, thus reducing the racetrack effect. The "y" region of the hyper-turn channel exhibits minimal tilting compared to the "y" region of the U-shaped turn channel. Serpentine channels with otimized taper designs operate at >90% separation efficiency of a straight channel of comparable effective length.
             

Microfabricated DNA Sequencer

The speed with which the human genome was completed was largely due to two breakthroughs: 1) Energy transfer labels and 2) capillary array electrophoresis instrumentation. Capillary array electrophoresis sequencers are now used exclusively in production genomics facilities, such as the DOE Joint Genome Institute, for new sequencing projects involving targets such as model organisms (Fugu) and a variety of microbes. Capillary-based sequencers are a robust DNA sequencing platform, but among other drawbacks, require 2-3 hours for analysis. Our lab has demonstrated that capillary electrophoresis microchips are capable of rapid DNA sequencing, but the readlength (the # of bases called to >99% accuracy) is restricted due to the length of the microchannel. Our earlier turn work circumvents this restriction with the introduction of serpentine channel geometries with hyper-turns. By incorporating the optimum turn geometry in a 96-lane microfabricated capillary array, an effective channel length of 15.9 cm was achieved. The device produced 41 kbp of >99% accuracy M13mp18 vector sequence in 25 min, a 5-fold improvement over conventional capillary array instrumentation.
                   

Left: The microfabricated capillary array electrophoresis chip design. A) The 96 lanes are grouped into 48 doublet structures on a 150-mm dia glass wafer. B) Each doublet structure contains two sample wells, linked to a common cathode and waste well. The 1.2-nL plug is formed at the intersection of the sample arm and waste arm and travels down the 15.9-cm serpentine channel, which contains four hyper-turns. C) The hyper-turn geometry used is a short symmetric taper with a radius of curvature of 250 um.

Right: The 96-lanes of DNA sequence in a pseudogel format. Of the 96 lanes, only one failed to produce sequence. The 41 kbp were collected in 24 minutes and exhibited excellent lane-to-lane reproducibility.
                   
References

Woolley, A. T. and Mathies, R. A. Proc. Natl. Acad. Sci. U. S. A. 91:11348-11352 (1994). [pdf]

Woolley, A. T. and Mathies, R. A. Ultra-High-Speed DNA Sequencing Using Capillary Electrophoresis Chips, Anal. Chem. 67: 3676-3680 (1995).

Woolley, A. T., Sensabaugh, G. F. and Mathies, and R. A. High-Speed DNA Genotyping Using Microfabricated Capillary Array Electrophoresis Chips, Anal. Chem. 69:2181-2186 (1997).
[Abstract] [Full Text in HTML] [Full Text in PDF]

Simpson, P. C., Roach, D., Woolley, A. T., Thorsen, T., Johnston, R., Sensabaugh, G. F. and Mathies, R. A. High Throughput Genetic Analysis using Microfabricated 96 Sample Capillary Array Electrophoresis Micro-Plates, Proc. Natl. Acad. Sci. U.S.A. 95: 2256-2261 (1998).
[Abstract] [Full Text in HTML] [Full Text in PDF]

Shi, Y. N., Simpson, P. C., Scherer, J. R., Wexler, D., Skibola, C., Smith, M. T., and Mathies, R. A. Radial Capillary Array Electrophoresis Microplate and Scanner for High-Performance Nucleic Acid Analysis, Anal. Chem., 71: 5354-5361 (1999).
[Abstract] [Full Text in HTML] [Full Text in PDF]

Paegel, B. M., Hutt, L. D., Simpson, P. C. and Mathies, R. A. Turn Geometries for Minimizing Band Broadening in Microfabricated Capillary Electrophoresis Channels, Anal. Chem. 72:3030-3037 (2000).
[Abstract] [Full Text in HTML] [Full Text in PDF]

Scherer J. R., Paegel B. M., Wedemayer G. J., Emrich C. A., Lo J., Medintz I. L., and Mathies R. A. High-Pressure Gel Loader for Capillary Array Electrophoresis Microplates, Biotechniques, 31: 1150-56 (2001).

Paegel, B. M., Emrich, C. A., Wedemayer, G. J., Scherer, J. R., and Mathies, R. A. High-Throughput DNA Sequencing with a Microfabricated 96-Lane Capillary Array Electrophoresis Bioprocessor, Proc. Natl. Acad. Sci. U. S. A., 99: 574-579 (2002).
[Abstract] [Full Text in HTML] [Full Text in PDF]

Paegel B.M., Yeung S.H.I. and Mathies R.A. Microchip Bioprocessor for Integrated Nanovolume Sample Purification and DNA Sequencing, Analytical Chemistry, 74, 5092-5098 (2002).
[Abstract] [Full Text in HTML] [Full Text in PDF]

Blazej R.G., Paegel B.M. and Mathies R.A. Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping, Genome Research, 13, 287-293 (2003).
[Abstract] [Full Text in HTML] [Full Text in PDF]

Paegel, B. M., Blazej, R. G., and Mathies, R. A. Microfluidic Devices for DNA Sequencing Sample Preparation and Electrophoresis Analysis, Current Opinions in Biotechnology,14, 42-50 (2003).
[Full Text in PDF]