The goal of this project is to develop a micro- and nanofluidic DNA sizing platform with a sensitivity, speed, and dynamic range that is unmatched by current sizing technologies. We find that fluorescence burst analysis of individual DNA molecules transported through sub-micrometer scale channels provides accurate and precise sizing of fragments between 100 bp and 10s of Mbp. In this single- molecule sizing platform, unlike in gel or capillary electrophoresis, the same instrument, device, and set of operating conditions produce optimal sizing performance across the full dynamic range. This capability in a low-cost, robust platform will be useful for many assays that require the measurement of DNA size distributions in heterogeneous solutions. Given the ability to rapidly size DNA molecules longer than 50 kbp, the platform is particularly valuable for the quality control of long-read sequencing libraries, improving the efficiency of sequencing workflows. Consumable fluidic devices that allow the precise control of DNA transport can be fabricated using injection molding of thermoplastics. In this project, we will define the optimal device designs and operating conditions to maximize the accuracy, precision, sensitivity, and dynamic range of this high-throughput platform. Once these parameters are defined in Phase 1, we will enhance the stability and reproducibility of sizing runs in Phase 2 of the project. This will be done through a combination of mechanical and optical engineering and the use of sizing markers. Sizing performance will be validated against pulsed-field gel electrophoresis. The project will culminate in a demonstration of platform utility in characterizing the quality of high-mass DNA before and after library preparation for long-read sequencing.
The goal of this project is to develop a micro- and nanofluidic DNA sizing platform with a sensitivity, speed, and dynamic range that is unmatched by current sizing technologies. Current technologies do not allow the sizing of DNA molecules larger than 20 kbp with a resolution or speed optimized for the quality control of long-read sequencing libraries. We have demonstrated a single-molecule nanofluidic sizing platform that can accurately size DNA fragments between 100 bp and 10s of Mbp and can improve sequencing workflows and the efficiency of genomic studies.