The long term objective of this proposal is to develop a fully integrated chip and reader capable of single molecule analysis of linearized, native state genomic material. The anticipated embodiment will permit direct visualization and analysis of megabase fragments of DNA extracted directly from a sample (possibly a single cell) with resolution better than 400 bp. Furthermore, the chip will accommodate massively parallel analyses of individual DNA molecules in a high-throughput manner thus providing statistically relevant data in a timely fashion. Our approach will focus on development of the nanochannel array, a device previously demonstrated by us to linearize single molecules of DNA in a massively parallel fashion. The device relies upon an array of channels with diameters less than 100 nm. As DNA is flowed through such channels, it becomes physically constrained and is forced to uncoil, thus permitting linear interrogation using fluorescent microscopy. Such analyses could serve to greatly enhance our understanding of genomic instabilities related to cancer and other genetic diseases. A critical consideration for the commercialization of this device is the design and manufacture of the nanochannels and supporting chip architecture. Currently, the device is composed of silicon and patterned using nanoimprint lithography (NIL). NIL is a low cost, high throughput nanomanufacturing process developed for the semiconductor industry as a means of further reducing the size of transistors in traditional integrated circuits. We have leveraged this technology for the manufacture of bio chips. Though well-suited for creating nanoscale features, further development of the process is required to accommodate a sample derived directly from the biologist's pipette. We hypothesize that the ideal device will require a transitional region from a macroscopic solution environment to the nanoscale channels. Furthermore, we expect that this transition is most economically developed by relying on conventional, plastic-based microfluidic manufacturing technologies for micron-scale features and silicon based approaches for the sub-micron transition leading to the channels themselves. Integration of these 2 manufacturing regimes is of critical importance. Completion of this project will result in a single, integrated biochip device capable of providing statistically significant, single molecule analysis of megabase DNA with 400 bp resolution. Such a device will be crucial to the future of single cell, single molecule analyses. Principal Investigator/Program Director (Last, First, Middle): Cao, Han Narrative: Single molecule analysis of long, genomic DNA will enable greater understanding and improved treatment of genetic diseases, especially cancer, by providing contextual information as to the nature of genetic polymorphisms. A standardized platform based on parallel nanochannels could serve as a basis for consistent, high-throughput genomic analyses in future patient care. ? ? ?
