Extraction of DNA from gels following electrophoresis is widely used to obtain purified size selected DNA fragments from mixtures. The DNA thus obtained is used for cloning and sequencing reactions. Gel extraction is part of next generation sequencing protocols that are becoming the primary source of genetic information. That information is widely used in translational studies and promises to be a cornerstone of ""personalized medicine"" where therapeutic courses are linked to an individual's genetic makeup. Rapid and simple nucleic purification products that improve workflow and reduce cost are vital to advances in genomics. Diffinity Genomics has licensed novel materials technology developed in the PI's lab at the University of Rochester that can be used for fast, inexpensive and simple biomolecular separations needed to purify nucleic acid reactions. The work in the present proposal involves adapting that technology and adding a new approach to selective capture of DNA from dissolved gel slices using silica particles functionalized with DNA intercalators. Our goal is to make products for rapid gel extraction using a three step pipette-based protocol. Current approaches to extraction of DNA from gels require multiple steps taking more than 10 minutes and use several reagents. Diffinity's purification methods are distinct because they use specially configured surfaces that selectively adsorb or repel desired solution components. This enables a process with fewer steps that can be implemented by retaining particles with the specially functionalized surfaces in pipette tips so that the purification process is reduced simply to aspirating the reaction solution and dispensing the purified DNA reaction solution. The approach based on functional pipettes is easily automated with liquid handlers for high throughput applications. We will demonstrate that we can configure silica particle surfaces appropriate to a three-step extraction of DNA from dissolved gel slices that take less than 3 minutes. If successful, in Phase II, we will integrate the particles into pipette tips designed to enable rapid mixing, verify the efficacy for applications to DNA sequencing and cloning, and exploit the manufacturing technology developed for a current product (RapidTipTM) to launch a gel extraction product.
Public Health Relevance: Cloning and sequencing of DNA are very important to medical research and diagnosis. Extracting DNA from gels following electrophoresis is widely used for cloning applications and is part of next generation sequencing protocols. We propose a new approach to extracting DNA from gels that will reduce the time;cost and labor associated with the process and be easily automated for high throughput applications.
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