Research Project
6074676
BBI BioSeq scientists have demonstrated that high hydrostatic pressure can be very useful in many steps in the nucleic acid sample preparation process including lysis of cells, deproteination, inhibition of ribonucleases and purification of nucleic acid from various biological samples. In the course of this Phase I study, we propose to examine the feasibility of using high pressure to lyse 'difficult cells', such as mycobacteria and animal tissues, such as liver. Parameters that will be evaluated include pressure, cycling conditions, temperature, and chemical additives. Release of nucleic acid from cells and binding and elution of these nucleic acids to solid phase matrices will be examined to establish the essential elements of a rapid extraction system which will be amenable to automation. The optimized sample extraction procedure will be rapid, require minimal sample handling, require no harsh chemical additions or withdrawals, and will result in high yield, high purity nucleic acids suitable for the immediate downstream processing. In the course of Phase II, a high throughput system for DNA and RNA isolation from a variety of biological samples will be developed and will include design of instrumentation, disposables and reagents required for efficient preparation of nucleic acids in a 96-well automated format. PROPOSED COMMERCIAL APPLICATION: The application of the high pressure cell lysis procedure proposed here is aimed to overcome the current bottleneck in the sample purifications and will greatly broaden the application of automated methods for nucleic acid analysis for a wide variety of high throughput applications. if successful, this approach will represent a revolutionary advancement in nucleic acid sample manipulations allowing preparation of up to 96 samples within a 30 minute period. It will benefit not only molecular diagnostics, but also fields, such as genomics, forensic analysis, genetic disease and cancer research.
