Cavitation Enhancement of Biospecimen processing for Improved DNA Fragmentation

描述(由申请人提供)：随机、无偏的DNA片段是下一代测序(NGS)和染色质免疫沉淀(CHIP)所必需的。由于DNA碎片对于NGS和芯片来说都可能是一个非常有问题的步骤，因此任何提高这一步骤的效率和一致性的技术都将是研究实验室和临床诊断中非常理想的。此外，一种可以使这一步骤变得更容易、不需要新设备、实验室成本很低的技术将是理想的。我们建议使用脂类包裹的微泡来从纯化的基因组和甲醛交联物样品中分离DNA。我们最近探索了这项技术的可行性，结果令人印象深刻。初步数据表明，微泡可以大大提高声学DNA片段的一致性。此外，这些气泡以微升的体积添加到DNA或细胞悬浮液中，每孔的成本从1美分到10美分不等，并且可以与任何标准的声学声谱仪一起使用，与其他改善DNA剪切的技术相比，大大节省了成本。此外，微泡技术大大减少了优化剪切所需的时间，潜在地极大地提高了该技术的吞吐量。作为第二个方面，我们还将评估微泡技术在加强福尔马林固定石蜡包埋(FFPE)组织处理或微活组织检查方面的潜力。我们的目标将是确定这一现象最佳表现的条件。测试变量包括缓冲试剂、微泡大小、微泡浓度、声频、声峰压力和超声持续时间。该项目最后将出版《标准作业程序》，以传播这项新技术的效用。 公共卫生相关性：下一代测序在了解与癌症相关的基因突变方面发挥着越来越重要的作用。在这项技术中，DNA片段是一个关键但有问题的步骤。我们的初步数据表明，我们有一种技术可以提高这项技术的稳健性，并通过在剪切悬浮液中应用声学活性微泡来减少时间和成本。我们将探索和优化这一新方法的各个方面。

DESCRIPTION (provided by applicant): Random, unbiased fragmentation of DNA is necessary for next-generation sequencing (NGS) and chromatin immunoprecipitation (ChIP). Since DNA fragmentation can be a very problematic step for both NGS and ChIP, any technology that increased the efficiency and consistency of this step will be highly desirable for both research laboratories and in clinical diagnostics. Also, a technology that could make this step easier with no new equipment and very little cost to the laboratory would be ideal. We propose to apply the use of lipid encapsulated microbubbles to the fragmentation of DNA from both purified genomic and formaldehyde crosslinked samples. We have recently explored the feasibility of this technology, and our results were impressive. Preliminary data indicate that microbubbles can greatly improve the consistency of acoustic DNA fragmentation. Additionally, these bubbles are added in microliter volumes to the DNA or cell suspension at a cost ranging from one to ten cents per well, and can be used with any standard acoustic sonicator, presenting substantial cost savings compared to other techniques to improve DNA shearing. Furthermore, the microbubble technique greatly reduces the time required to optimize shearing, potentially greatly improving the throughput of this technique. As a second aspect, we will also assess the potential of microbubble technology to enhance tissue processing of formalin-fixed paraffin embedded (FFPE), or microbiopsies. Our goal will be to determine conditions for optimal performance of this phenomenon. Variables tested will include buffer reagents, microbubble size, microbubble concentration, acoustic frequency, acoustic peak pressure, and sonication duration. The project will conclude with publication of Standard Operating Procedures to disseminate the utility of this new technology. PUBLIC HEALTH RELEVANCE: Next-generation sequencing is playing an increasingly important role in understanding genetic mutations associated with cancer. DNA fragmentation is a crucial, but problematic step in this technique. Our preliminary data suggests that we have a technology to improve the robustness of this technique, as well as reduce time and cost through the application of acoustically active microbubbles to the shearing suspension. We will explore and optimize aspects of this novel approach.