EXTRACTION OF NUCLEIC ACIDS FROM CELLS USING PRESSURE

BBI BioSeq科学家已经证明，高静水压力在核酸样品制备过程的许多步骤中都非常有用，包括细胞裂解、脱蛋白、抑制核糖核酸酶和从各种生物样品中纯化核酸。在这个一期研究过程中，我们建议研究使用高压裂解“困难细胞”的可行性，如分枝杆菌和动物组织，如肝脏。评估的参数包括压力、循环条件、温度和化学添加剂。将研究从细胞中释放核酸以及这些核酸与固相基质的结合和洗脱，以建立可自动化的快速提取系统的基本要素。优化的样品提取程序将快速，需要最少的样品处理，不需要苛刻的化学添加或提取，并将产生高产量，高纯度的核酸，适合直接下游处理。在第二阶段的过程中，将开发一种高通量系统，用于从各种生物样品中分离DNA和RNA，并将包括在96孔自动化格式中有效制备核酸所需的仪器，一次性和试剂的设计。建议的商业应用：这里提出的高压细胞裂解程序的应用旨在克服样品纯化中的当前瓶颈，并将大大拓宽自动化核酸分析方法的应用范围，以实现各种高通量应用。如果成功，这种方法将代表核酸样品操作的革命性进步，允许在30分钟内制备多达96个样品。它不仅有利于分子诊断，而且有利于基因组学、法医分析、遗传疾病和癌症研究等领域。

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.