As new genetic analysis methods are developed, rising emphasis is being placed on high quality sample preparation and the ability to obtain large quantities of high molecular weight and high purity DNA. In DNA sequencing, 3rd generation technologies offer long read lengths up to 30 kb and require high quality isolated DNA. In gene therapy and DNA vaccines, high binding capacity could be extremely useful in isolating industrial quantities of long plasmids. In genetic analysis of extremely rare sequences or functional analysis of large chromosomal rearrangements, high molecular weight input DNA reduces the chance that fragmentation will occur in a region of interest. In Phase I, we created a novel thermoplastic, silica substrate, containing a hierarchical layering of microscale folds and nanoscale silica lamella called Nanobind. Through the combined effects of minimized shear force exposure and high surface area, we are able to extract vast amounts (10- 100X more) of high molecular weight genomic DNA (>100 kb) using a simple bind, wash, and elute protocol that is both fast (<45 min) and inexpensive ($0.01 per substrate). In this Phase II SBIR, we will develop Nanobind technologies to address 3 areas where critical limitations exist with current extraction methods. First, we will create a facile method for high MW extraction by developing a magnetic Nanobind substrate. Second, current automated extraction systems rely exclusively on beads and columns and tend to fragment DNA to <50 kb. We will develop MagNanobind HTP, a 96-well format for high-throughput, high MW DNA extraction that is compatible with a variety of existing automated extraction instruments. Third, we will develop a microvolume Nanobind cartridge to perform small volume extractions using clinical samples <1 L or <10 ng. Finally, we will validate the Nanobind DNA/RNA extraction platform by comparing against microparticle, spin column, and precipitation methods using 3 common genetic and epigenetic assays. Nanobind could immediately impact the DNA extraction market as it uniquely combines the speed and ease of microparticles and spin columns with performance of phenol-chloroform.
As new genetic analysis methods are developed, rising emphasis is being placed on high quality sample preparation. This project will develop a new silica nanomaterial for nucleic acid isolation that is fast and easy yet results in large quantitie of high quality DNA and RNA. Such a method could greatly reduce the cost of sample preparation while improving the quality of data from genetic research.
| Zhang, Ye; Zhang, Yi; Burke, Jeffrey M et al. (2016) A Simple Thermoplastic Substrate Containing Hierarchical Silica Lamellae for High-Molecular-Weight DNA Extraction. Adv Mater 28:10630-10636 |
| Nguyen, Trung C; Cruz, Miguel A; Carcillo, Joseph A (2015) Thrombocytopenia-Associated Multiple Organ Failure and Acute Kidney Injury. Crit Care Clin 31:661-74 |
