SBIR-STTR Award

The objective of the proposed study is to develop new materials for the rapid isolation of nucleic \ acids from complex matrices such as whole blood. In Phase I we will prepare functionalized \ microparticles for binding and releasing nucleic acids that utilize proprietary chemistry recently i developed at Lumigen. These materials find many uses but are intended particularly for the rapid ' and simplified isolation of genomic nucleic acids from unpurified or heterogeneous matrices, especially DNA in mammalian tissue or whole blood. This is made possible by a unique feature of these materials: their ability to capture DNA and/or RNA under virtually any reasonable conditions. As a result we have been able to bind DNA from whole blood without any lysis step. The prototype new materials bind nucleic acids with exceptional strength under a range of conditions and resist release under a wide variety of conditions. The unique binding properties of the new materials will be utilized to develop optimized methods for stringently washing and releasing the bound nucleic acid in a highly controllable fashion. Preliminary experiments have demonstrated that a cleavable link between the solid support and the binding moiety can be controllably severed to release nucleic acid back into solution. Nucleic acids purified with first generation materials have been found compatible with downstream applications including amplification technologies such as PCR and LMO, a ligase-based nucleic acid amplification technology invented by the PL We expect to produce one or more general use kits for isolating DNA from whole blood without any lysis step and in a quantity and quality acceptable for use in downstream amplification and diagnostic applications. For the initial phase of the project we expect to develop both non-magnetic and | magnetically responsive microparticles from different solid phase support materials. Since the j prototype new materials have shown exceptionally strong binding to nucleic acids, we plan further to begin exploring the use of the developed materials in capturing nucleic acids from low abundance viral and bacterial infectious agents from samples such as blood and other body fluids. This work serves as a prelude to the development of nucleic acid-based test methods for pathogens in human blood and food products to be undertaken in Phase n

This Phase II project continues the development and application of new materials, especially magnetic microparticles for directly isolating DNA from cells, tissues, and biological fluids, including whole blood, in an extremely rapid format. The materials eliminate the need to perform a lysis step and capture DNA from complex materials thereby simplifying sample preparation methods. The functionalized microparticles for binding and releasing nucleic acids utilize a proprietary chemistry which enables them to spontaneously and rapidly lyse cells and capture the exposed DNA in situ under virtually any biological conditions. As a result DNA can be isolated from whole blood without any lysis step. The unique surface phosphonium group chemistry causes nucleic acids to bind with exceptional strength until released by a unique cleavage mechanism where a linker group is selectively broken by a simple chemical reaction in order to free DNA from the solid surface. Release by chemical cleavage rather than traditional elution prevents premature, uncontrolled release of material and yields pure nucleic acid. Isolated DNA is compatible with downstream applications such as PCR. Additional linking groups and cleavage methods are being explored. Phase II will develop the expertise to perform the complete synthesis including preparing the magnetic cores, coating the cores, and functionalizing with linker and head groups. The capability to manufacture the fuctionalized magnetic particles on a large scale will be developed. We have produced a prototype kit for isolating DNA from whole blood. We areing the range of applications of these new materials is being expanded to plasmid purification, isolation of DNA from viruses, and capture of DNA from infectious microorganisms in urine, foodstuffs and culture media with no further isolation steps. The materials are expected to streamline sample preparation and DNA extraction across a broad variety of applications. - This Phase II project continues the development and application of new materials, especially magnetic microparticles for directly isolating DNA from cells, tissues, and biological fluids, including whole blood, in an extremely rapid format. The materials eliminate the need to perform a lysis step and capture DNA from complex matrices thereby simplifying sample preparation methods. The materials will find use in a great number of applications in the life sciences and diagnostics industries for which DNA extraction is necessary.

Thesaurus Terms:
blood test, magnetic field, method development, microprocessor /microchip, nucleic acid purification chemical cleavage, chemical stability, particle, phosphorus compound, reagent /indicator, surface coating biotechnology, high throughput technology, polymerase chain reaction