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.
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.
| Kasoji, Sandeep K; Pattenden, Samantha G; Malc, Ewa P et al. (2015) Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath. PLoS One 10:e0133014 |
