The Human Genome Project is a high priority area for the NIH, since the long-range goals of this project will have important ramifications in ameliorating genetic diseases, improving health care and possibly altering the genetic makeup of an individual. In order to tackle sequencing on a genomic scale, significant strides must be made in technologies which will acquire raw sequence data on an appropriate time-scale. The proposed research is directed toward the development of a highly multiplexed DNA sequencing device using a single lane, single fluor approach and capable of handling large-scale sequencing projects. The base identification strategy presented in this application uses a series of four recently synthesized near-IR (NIR) fluorescent dyes which contain a different internal heavy-atom (halogen) situated on a remote site in the molecular framework of the chromophore. The principal advantage of using NIR dyes is that detection can be performed directly in the gel column without sacrificing detection sensitivity. In addition, NIR fluorescence allows the use of simple diode lasers and solid-state photodetectors. The heavy- atom modified NIR dyes possess similar absorption and emission maxima, therefore requiring only a single laser for excitation and a single detection channel. However, these dyes show incremental changes in their fluorescence lifetimes, ranging from 820-900 ps. Base identification will be affected by performing dynamic measurements during capillary gel electrophoresis using simple algorithms to calculate the lifetime. The advantages of lifetime measurements for ddNTP identification is that the measured value is insensitive to the amount of material comprising each electrophoretic band. Therefore, the proposed single lane, single fluor method will allow the use of dye-labeled ddNTPs and, in addition, a variety of different polymerase enzymes can be used to fit the particular application without being restricted to the use of the modified T7 DNA enzyme. Multiplexing is accomplished using a series of single mode optical fibers for light delivery and collection, which direct the emission onto a multichannel detector comprised of single photon avalanche diodes. The capillary array is constructed on a rigid glass plate with microchannels photolithographically etched onto it. The light delivery and collection optical fibers are permanently mounted on this plate and placed at the gel interface, forming one wall of the separation channel. The glass plate has conventional fused silica capillaries fixed on either end in order to permit easy placement in the anodic and cathodic reservoirs. The sieving medium consists of a low viscosity linear polyacrylamide, which can be replaced once breakdown has occurred due to extended high voltage operation. With this arrangement, the sequencing device can be run continuously without requiring column removal or optical realignment. In its final form, the device will possess the capability to run 100 separation lanes or more simultaneously using a single laser for excitation and a single detection channel for processing the sequencing data.
| Ford, S M; Davies, J; Kar, B et al. (1999) Micromachining in plastics using X-ray lithography for the fabrication of microelectrophoresis devices. J Biomech Eng 121:13-21 |
