Many important variations in the genomes between different individuals in a population are in the kilobase pair (kbp) to megabase pair range. As they often feature numerous repeat sequences and inversions, these variations are difficult to analyze using next generation DNA sequencing, and their length prevents using hybridization microarrays. DNA barcoding, where genomic information is read from single molecules of stretched DNA, is emerging as a key tool for high-throughput detection of such large-scale rearrangements. Nanochannels present an especially attractive approach to reading DNA barcodes; when the labeled DNA is injected into a nanochannel, it stretches out and fluctuates about its equilibrium extension. Nanochannels reduce the error in the measurement of the genomic distance between barcodes by sampling the statistically independent configurations resulting from these fluctuations. While there are theories for the extension and dynamics of DNA in very small (< 20 nm) and relatively large (> 500 nm) nanochannels, these theories are of little use for device engineering - the small channels are difficult to fabricate and operate, whereas the larger channels do not provide enough stretching to resolve the barcodes. As a result, most devices operate between these two limits. The absence of any fundamental understanding of the behavior of DNA in channels from 100-500 nm in width is hindering the engineering and optimization of nanochannels used for DNA barcoding and proposed uses of nanochannel arrays as a platform for next generation sequencing.
Specific Aim 1 will build upon substantial preliminary data to produce an experimentally validated model of DNA in confinement. Accomplishing this goal requires a tight integration of Monte Carlo simulations, Brownian dynamics simulations with fluctuating hydrodynamic interactions, nanofabrication and fluorescence videomicroscopy.
In Specific Aim 2, this model will be used in conjunction with experiments on barcoded DNA to (i) optimize the resolution of the state-of-the-art protocol and (ii) test new, model-based protocols that should offer substantial advantages in cost and analysis time. This research is significant because it will greatly advance the understanding of confined DNA, in particular the dynamics of the chain. By resolving the open scientific questions, this research will advance DNA nanochannel array technologies for genomics. This technology focus is enhanced by a collaboration with BioNano Genomics. Accomplishing the specific aims requires an innovative synergistic coupling between different simulation methods and experimental techniques and partnership with industry. This work will impact the transition of nascent nanochannel technology to end-users in the genomics community through a rational engineering framework for optimizing barcode reading protocols. It is expected that the novel model-based measuring protocol produced from this research will lead to hundred-fold improvements in the analysis time while reducing the cost of the nanochannels, associated hardware and consumables.
The proposed research is relevant to public health because it will improve the accuracy, analysis time, and cost of the devices required for large-scale analysis of human genomes and the genomes of pathogens, in particular those features that are not amenable to analysis by high throughput sequencing or microarrays. The proposed research is relevant to the mission of the NHGRI to develop and improve novel technologies for genome research.
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