Cavitation Enhancement of Biospecimen processing for Improved DNA Fragmentation

生物样本处理的空化增强以改善 DNA 断裂

• 批准号: 8432313
• 负责人: Paul A Dayton
• 金额: $ 19.84万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-12 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432313
• 关键词: Acoustics; Base Pairing; Biology; Biopsy; Buffers; Cancer Diagnostics; Cancer Patient; Cells; Chromatin; Clinical; Companions; Contrast Media; Cost Savings; Cytolysis; DNA; DNA Fragmentation; DNA-protein crosslink; Data; Diagnosis; Diagnostic; Diagnostic tests; Encapsulated; Engineering; Epidermal Growth Factor Receptor; Epigenetic Process; Equipment; Erlotinib; Formaldehyde; Formalin; Frequencies; Future; Gefitinib; Gene Expression; Gene Mutation; Genome; Genomics; Goals; Individual; Laboratories; Laboratory Research; Legal patent; Length; Lipids; Literature; Malignant Neoplasms; Mediating; Medicine; Methods; Microbubbles; Mutation; Non-Small-Cell Lung Carcinoma; Normal Cell; Paraffin Embedding; Patients; Performance; Pharmaceutical Preparations; Physics; Play; Procedures; Process; Proteins; Protocols documentation; Publications; Publishing; Reading; Reagent; Reproducibility; Research; Role; Sampling; Sequence Analysis; Sonication; Specimen; Suspension substance; Suspensions; Techniques; Technology; Testing; Time; Tissues; Tube; Tumor-Associated Process; Ultrasonography; anticancer research; assay development; base; cancer cell; cancer therapy; cell fixing; chromatin immunoprecipitation; cost; crosslink; falls; genetic profiling; improved; innovation; interdisciplinary collaboration; metaplastic cell transformation; neoplastic cell; new technology; next generation; novel strategies; pressure; tissue processing; tool; tumor

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.

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片段化对于NGS和ChIP来说都是一个非常有问题的步骤，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非常需要的。此外，如果有一种技术可以使这一步变得更容易，不需要新的设备，而且实验室的成本也很低，那将是理想的。我们建议将脂质包封微泡应用于纯化基因组和甲醛交联样品的DNA片段化。我们最近探索了这项技术的可行性，我们的结果令人印象深刻。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加入到DNA或细胞悬浮液中，每孔的成本在1到10美分之间，可以与任何标准的声学检测器一起使用，与其他技术相比，大大节省了成本，以改善DNA剪切。此外，微泡技术大大减少了优化剪切所需的时间，潜在地大大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）组织处理或显微活检方面的潜力。我们的目标是确定这种现象的最佳性能条件。测试的变量将包括缓冲试剂，微泡大小，微泡浓度，声波频率，声峰值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用途。