EXTRACTION OF NUCLEIC ACIDS FROM CELLS USING PRESSURE

BBI BioSeq的科学家已经证明，在核酸样品制备过程中的许多步骤中，高静压都非常有用，包括细胞裂解、去蛋白、抑制核糖核酸酶和从各种生物样品中提纯核酸。在这项第一阶段的研究过程中，我们建议研究使用高压来裂解‘困难细胞’的可行性，如分枝杆菌和动物组织，如肝脏。将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将这些核酸结合和洗脱到固相基质中，以建立易于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是快速的，需要最少的样品处理，不需要苛刻的化学添加或撤回，并将产生适合直接下游加工的高产率、高纯度的核酸。在第二阶段期间，将开发一个从各种生物样本中分离DNA和RNA的高通量系统，其中将包括设计有效地以96孔自动格式制备核酸所需的仪器、一次性用品和试剂。拟议的商业应用：这里提出的高压细胞裂解程序的应用旨在克服目前样品纯化方面的瓶颈，并将极大地拓宽核酸分析自动化方法的应用范围，用于各种高通量应用。如果成功，这种方法将代表着核酸样本处理方面的革命性进步，允许在30分钟内制备多达96个样本。它不仅对分子诊断学有利，而且对基因组学、法医分析、遗传病和癌症研究等领域也有好处。

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.