High Pressure Sample Preparation Instrumentation for DNA Sequencing.

描述(由申请者提供)：在NIH挑战实现1000美元基因组的鼓舞下，目前大规模并行测序(MPS)的发展继续增加产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产量和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速度。这项建议的重点是论证在受控和高能条件下利用微型(&lt；200？L样本)超高压(高达400 Mpa)单程放电产生输入样本用于未来大规模并行测序(MPS)的可行性。所提出的系统建立在已建立的流体剪切在高差压喷嘴流动下产生的流体动力学机制的基础上。由于强烈的粘性阻力，穿过喷嘴高速度梯度的长DNA片段被拉开。例如，在这些高压下，流体的流动速度将在很短的过渡距离内达到音速的3倍。通过改变压力、流量、背压等控制参数，可以实现不同程度的破碎。这种裂解过程不是概率的，它作用于通过喷嘴的每一个DNA分子。第一阶段建议的重点是优化控制参数，以实现所需的DNA碎片性能，这是在本文所述的两种喷嘴设计替代方法的几个原型上已经完成的初步工作的后续工作。这些方法中的每一种都可以被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。

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.