DOE Human Genome Program Contractor-Grantee
38. DNA and Protein Analyses on Microfabricated Devices
R. S. Foote, Y. Khandurina, I. M. Lazar, Y. Liu, T. McKnight, L. C. Waters, S. C. Jacobson, R. S. Ramsey, and J. M. Ramsey
Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6142
Microfabricated, microfluidic devices are being developed for both nucleic acid and protein analyses. An integrated system for rapid PCR-based analysis on a microchip has been demonstrated. The system couples a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel electrophoresis platform. This configuration allows fast (~ 1 min/cycle) and efficient DNA amplification on-chip followed by electro-phoretic sizing and detection on the same chip. On-chip DNA concentration has been incorporated into the system to further reduce analysis time by decreasing the number of thermal cycles required. The concentration-injection scheme enables detection of PCR products after performing as few as 10 thermal cycles with a total analysis time under 20 min, with a starting template copy number of fewer than 15 molecules per injection volume.
Electrophoretic separations of proteins have been carried out on microchips with on-chip, post-column labeling for detection by laser-induced fluorescence. The post-column labeling format avoids peak broadening and loss of resolution due to heterogeneous product formation in prelabeling reactions. Two-dimensional separations of tryptic peptides were demonstrated on microchips that combine micellar electrokinetic chromatography (MEKC) and high-speed capillary electrophoresis (CE). Effluent from the first dimension is sampled onto the second dimension every few seconds and the entire analysis is completed within 10 minutes. Structures that incorporate an electrospray element have also been devised and sub-attomole sensitivity demonstrated for peptide samples on a time-of-flight (TOF) mass analyzer. Proteolytic digestions with trypsin can be performed directly on the chip and the peptide fragments analyzed by TOFMS for protein identification. Tryptic peptides could be generated in less than 10 min for analysis of femtomol or subfemtomol amounts of protein.
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