) Advances in molecular biological techniques have provided a molecular basis for the diagnosis of many diseases and spawned the era of molecular diagnostics for cancer diagnosis and prognosis. The main methods associated with molecular diagnostics, polymerase chain reaction (PCR) for amplification of DNA and electrophoretic separation for detection of the amplified products, are labor-intensive, time- consuming and not amenable to automation. This methodological """"""""bottleneck"""""""" can be solved, in part, by capillary electrophoresis (CE) and more comprehensively by """"""""chip electrophoresis"""""""". Chip electrophoresis involves carrying out CE separations in micron-scale channels etched in a planar glass surface. In addition Co allowing for rapid separations as a result of miniaturization, the """"""""chips"""""""" also provide a convenient platform for integrating pre-electrophoresis sample processing. Consequently, sample preparation, PCR amplification and electrophoretic separation/detection can be amalgamated into a single microanalysis device. As part of the development of an integrated electrophoretic chip for cancer diagnostics, we plan to develop a chip-amenable approach to thermocycling. Using a tungsten lamp as an infrared heat source, noncontact temperature cycling will be executed on focused areas of the chip with temperature control accomplished through remote sensing. This will involve either the development of an IR sensing device (pyrometer) or a refractive index detection scheme, both of which will provide remote control of cycling. Since it will be important to automate the extraction of DNA from samples for on-chip PCR, we also plan to develop a chip-amenable methodology for purifying DNA from blood for on-chip PCR and electrophoresis. The focus of this proposal will be to explicit solid phase extraction methodologies for purification of DNA from white blood cells in a manner that can easily be extrapolated to the chip. Proof of feasibility for on-chip DNA purification. amplification and detection of cancer-specific DNA markers will be accomplished using acute myelogenous leukemia and B-cell lymphoma as model systems.
| Breadmore, Michael C; Wolfe, Kelley A; Arcibal, Imee G et al. (2003) Microchip-based purification of DNA from biological samples. Anal Chem 75:1880-6 |
| Wolfe, Kelley A; Breadmore, Michael C; Ferrance, Jerome P et al. (2002) Toward a microchip-based solid-phase extraction method for isolation of nucleic acids. Electrophoresis 23:727-33 |
| Tian, H; Brody, L C; Fan, S et al. (2001) Capillary and microchip electrophoresis for rapid detection of known mutations by combining allele-specific DNA amplification with heteroduplex analysis. Clin Chem 47:173-85 |
| Tian, H; Huhmer, A F; Landers, J P (2000) Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format. Anal Biochem 283:175-91 |
| Munro, N J; Snow, K; Kant, J A et al. (1999) Molecular diagnostics on microfabricated electrophoretic devices: from slab gel- to capillary- to microchip-based assays for T- and B-cell lymphoproliferative disorders. Clin Chem 45:1906-17 |
