Information from next generation sequencing 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. Extraction of DNA from gels following electrophoresis to obtain size-selected DNA fragments is a labor intensive part of library preparation present in the protocols of all of the major next generation sequencing platforms. Recently, Sage Science introduced a disruptive product for DNA size selection that electrically "switches" the desired DNA fragments out of the gel and obviates the need for physically extracting the DNA. Due to its speed and labor saving protocol, the Sage Science Pippin Prep is being quickly adopted by the rapidly growing next generation sequencing market. Unfortunately, with the Pippin Prep it remains necessary to do a relatively labor-intensive purification to remove the intercalating dyes used to identify the bands because they interfere with subsequent processes in next generation sequencing library preparation. Diffinity Genomics proposes to develop a single-step purification process based on a functional pipette tip that will eliminate intercalating dyes in less than 60 seconds while retaining the desired DNA. 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. Particle surfaces are specially configured to selectively adsorb or repel various solution components making it possible to retain desired components in solution while removing unwanted impurities. Diffinity has utilized that differential adsorption technology to launch a fast PCR purification product (RapidTipTM) that enables single-step PCR cleanup in less than a minute simply by aspirating the PCR product into a functional pipette tip and dispensing purified DNA reaction solution. The work in the present proposal is to develop particles with surfaces that selectively bind the intercalating dyes used by Sage Science and to make the particles easily dispersible in water to facilitate rapid mixing and collection of impurities. We will also ascertain whether the size-selected DNA obtained with the Sage Science instrument and Diffinity purification is suitable for next generation sequencing library preparation.
Public Health Relevance: Next generation sequencing of DNA is growing rapidly since it provides genetic information important to medical research and diagnosis. Size selection of DNA using gel electrophoresis is part of next generation sequencing protocols and a new product from Sage Science is streamlining that process. We propose a companion product that will reduce the time, cost and material waste associated with purifying the size-selected DNA obtained from the Sage Science process.
Public Health Relevance Statement: Next generation sequencing of DNA is growing rapidly since it provides genetic information important to medical research and diagnosis. Size selection of DNA using gel electrophoresis is part of next generation sequencing protocols and a new product from Sage Science is streamlining that process. We propose a companion product that will reduce the time, cost and material waste associated with purifying the size-selected DNA obtained from the Sage Science process.
NIH Spending Category: Biotechnology; Genetics; Human Genome
Project Terms: Address; Adopted; Adsorption; Agitation; Aspirate substance; base; Binding (Molecular Function); Biochemistry; Buffers; Caring; Chemistry; Collection; Companions; cost; Diagnosis; Diagnostic; DNA; DNA Sequence; DNA-Directed DNA Polymerase; drug development; ds-DNA; Dyes; Electrophoresis; Excision; Fluorescence; Gel; gel electrophoresis; Genetic; Genomics; Homidium Bromide; improved; Individual; infrared spectroscopy; instrument; instrumentation; Intercalating Agents; Left; Libraries; Licensing; Link; Location; Marketing; Measures; Medical; Medical Research; Medicine; Metric; next generation; novel; Nucleic Acids; One-Step dentin bonding system; Oranges; particle; Polymerase Chain Reaction; Preparation; Procedures; Process; Protocols documentation; Reaction; Sampling; Savings; Science; Sepharose; Silicon Dioxide; silochrome; Slice; Solutions; Speed (motion); success; Surface; System; Technology; Therapeutic; Time; translational study; trend; Triazines; Tube; Universities; Validation; wasting; Water; water solubility; Work
