Anatomic 3D synthetic tissue printer

DESCRIPTION (provided by applicant): Nanopores are emerging technologies that offer the prospect of long-read (gt100 kb) next-generation sequencing without the need for sample amplification. Such technology can complement existing short-read and high throughput sequencing platforms to reduce errors in de novo genome assembly and structural variant analysis, or entirely replace this technology if comparable error rates can be achieved; in either case, nanopores are positioned to make a considerable impact inthe growing application of genomics in medicine. A long-standing challenge for nanopore sequencing has been to develop a method for controlling the rate of DNA through the pore to ensure accurate sequencing during nucleotide sensing. While leading research and commercial methods address this by using enzymes on top of each pore to control DNA motion through the pore, our patented method of DNA control eliminates the need for enzymes or chemistry, offering a considerable reduction in cost and instrument complexity. Our patented method involves the use of two nanopores to capture and control each DNA molecule. Independent voltage control across each pore permits electrophoretic tug-of-war to pull the DNA in competing directions, and thereby control the rateand direction of each DNA through the pores during sensing. Phase I funding will develop a prototype dual-nanopore device to demonstrate capture and rate control of individual dsDNA through both pores, and mapping of grosser features (i.e., binding proteins) using the two ionic nanopore current measurements. The long-term objective is to couple the control method with a single-nucleotide sensor for a reusable and chemistry-free platform for sequencing long single-stranded DNA. There are three aims: Aim 1.Develop a dual-pore microfluidic chip and housing. The proposed work leverages expertise in the fabrication of high performance nanopore-bearing membranes and devices. The chip design minimizes access resistances, which is critical to preserving sensing during control. Pores are sufficiently close (200 nm) for dual- pore capture of gt1 kbp dsDNA, and sized (20-30 nm diam) for current sensing of dsDNA and bound proteins. Aim 2. Demonstrate capture and control of individual dsDNA. Preliminary analysis provides conditions under which the two ionic currents can sense DNA in each pore, and supports that the likelihood of second-pore capture following first-pore capture is high for the proposed geometry. Demonstrations of competing voltage control following capture will next be established, leveraging our expertise in voltage-control design. Aim 3. Demonstrate detection and localization of proteins bound to a single dsDNA molecule (2-50 kbp), achieving single protein resolution. We will build on the precedent for detecting RecA filaments formed on dsDNA using single nanopore devices, and also map the presence of phage lambda repressor which binds to specific sequences. The demonstrations support that the method has immediate commercial relevance, since mapping individual proteins (or, comparably, bound particle labels) on long dsDNA can be used for genome mapping, and without cameras or high resolution imaging methods. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: The proposed instrument will advance DNA sequencing and analysis by providing a new method to control DNA motion through nanopores that is entirely electrophoretic and does not require enzymes, chemistry or any auxiliary means of manipulating an attachment to the DNA. The proposed dual-pore device will have immediate use for genome mapping applications, e.g., to assist in de novo sequence assembly. Ultimately, the method will be coupled with a single-nucleotide sensor for direct genome sequencing of long single-stranded DNA.