High Pressure Sample Preparation Instrumentation for DNA Sequencing.

用于 DNA 测序的高压样品制备仪器。

• 批准号: 8454322
• 负责人: Alexander V Lazarev
• 金额: $ 14.11万
• 依托单位: PRESSURE BIOSCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454322
• 关键词: Address; Adoption; Animal Model; Back; Biomedical Research; Cells; Chemicals; Clinical; Confined Spaces; Cultured Cells; Cytolysis; DNA; DNA Damage; DNA Fragmentation; DNA Sequence; Development; Devices; Diagnostic; Elasticity; Ensure; Epigenetic Process; Equilibrium; Future; Genome; Genomics; Health Services Research; Human Microbiome; Industry; Lead; Length; Liquid substance; Methodology; Methods; Organism; Performance; Phase; Polymers; Preparation; Process; Property; Robotics; Role; Sample Size; Sampling; Silicones; Solutions; Speed; System; Systems Development; Techniques; Technology; Temperature; Testing; Time; Travel; Tube; Viscosity; Work; Yeasts; base; cost; design; experience; fluid flow; genome sequencing; improved; instrumentation; microbiome; miniaturize; next generation sequencing; parallel processing; pressure; prevent; prototype; public health relevance; scale up; seal; sound

DESCRIPTION (provided by applicant): Encouraged by NIH's challenge to enable the $1000 genome, current developments in massively parallel sequencing (MPS) continue to increase throughput and reduce the cost of whole-genome sequencing. Unfortunately, the corresponding sample preparation methodologies now lag behind in throughput and DNA quality, thus slowing the pace of discovery and adoption of sequencing techniques in clinical diagnostics. This proposal's focus is to demonstrate the feasibility of controlled DNA shearing utilizing miniature (<200¿L sample) very high pressure (up to 400Mpa) single pass discharge under controlled and highly energetic conditions to generate the input sample for future massively parallel sequencing (MPS). The proposed systems build on established hydrodynamic mechanism of fluid shear generated under high differential pressure nozzle flow. Long DNA fragments traversing though the nozzle's high velocity gradient are pulled apart due to intense viscous drag forces. For example, at these high pressures, fluid flow velocity will reach 3 times the speed of sound within a very short transition distance. By varying the control parameters of pressure and flow, back pressure, different levels of fragmentation should be achieved. This fragmentation process is not probabilistic and acts upon every DNA molecule that transits the nozzle. The Phase I proposal is focuses on the optimization of control parameters to achieve desired DNA fragmentation performance, following up on preliminary work already done on several prototypes of the two alternative approaches to nozzle design described herein. Each of these approaches can be multiplexed for high throughput use. During Phase II, we intend to expand our studies into large parallel process demonstration.

描述（由申请人提供）：在NIH挑战1000美元基因组的鼓舞下，大规模并行测序（MPS）的当前发展继续增加通量并降低全基因组测序的成本。不幸的是，相应的样品制备方法现在在通量和DNA质量方面落后，从而减缓了测序技术在临床诊断中的发现和采用的步伐。该提案的重点是证明在受控和高能量条件下利用微型（<200 L样品）非常高的压力（高达400 Mpa）单程放电进行受控DNA剪切的可行性，以生成用于未来大规模并行测序（MPS）的输入样品。所提出的系统建立在高压差喷嘴流下产生的流体剪切的流体动力学机制上。穿过喷嘴的高速度梯度的长DNA片段由于强烈的粘性阻力而被拉开。例如，在这些高压下，流体流速将在非常短的过渡距离内达到声速的3倍。通过改变压力和流量、背压的控制参数，应实现不同的破碎水平。这种断裂过程不是随机的，而是作用于通过喷嘴的每个DNA分子。第一阶段的建议是集中在控制参数的优化，以实现所需的DNA片段化性能，后续的初步工作已经完成了几个原型的两种替代方法，喷嘴设计在此描述。这些方法中的每一种都可以多路复用以用于高吞吐量使用。在第二阶段，我们打算将我们的研究扩展到大型并行过程演示。