Yuan Guoliang, Salalila Aljon, Hwang Sungjoo, Deng Zhiqun Daniel, Deng Shuang
Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, United States.
Department of Naval Architecture and Marine Engineering, University of Michigan, Ann Arbor, MI, United States.
Front Bioeng Biotechnol. 2025 Mar 25;13:1547909. doi: 10.3389/fbioe.2025.1547909. eCollection 2025.
High-throughput PCR screening is vital in synthetic biology and metabolic engineering, enabling rapid and precise analysis of genetic modifications. However, current methods face challenges including inefficient DNA extraction, high variability across sample types, scalability limitations, and the high cost of template DNA extraction. To address these common challenges, we developed a High-Throughput Genome Releaser (HTGR). This innovative device utilizes a squash-based method for rapid, cost-effective, and efficient DNA extraction, optimized for subsequent PCR reactions. After testing various synthetic materials, we selected a plastic that closely mimics the smooth surface and compression properties of microscope slides, ensuring reliable and consistent performance. The device comprises a 96-well plate and a Shear Applicator, designed for both manual and automated operation, and is compatible with standard liquid-handling robotic platforms. This compatibility simplifies integration into high-throughput PCR workflows. Additionally, we developed software to support its automated functions. Our results demonstrated that the specially engineered 96-well plate and HTGR effectively squash fungal spores, releasing sufficient genomic DNA for PCR screening with 100% efficiency. The genome releaser enables the preparation of PCR-ready genomic DNA from 96 samples within minutes, eliminating the need for an extraction buffer. Its adaptability to a wide range of microorganisms and cell types makes it a versatile tool that could significantly advance biomanufacturing processes.
高通量PCR筛选在合成生物学和代谢工程中至关重要，能够对基因修饰进行快速而精确的分析。然而，当前的方法面临着诸多挑战，包括DNA提取效率低下、不同样本类型间差异大、可扩展性受限以及模板DNA提取成本高昂。为应对这些常见挑战，我们开发了一种高通量基因组释放器（HTGR）。这种创新设备采用基于挤压的方法，可快速、经济高效地提取DNA，并针对后续PCR反应进行了优化。在测试了各种合成材料后，我们选择了一种能紧密模拟载玻片光滑表面和压缩特性的塑料材料，以确保可靠且一致的性能。该设备包括一个96孔板和一个剪切施加器，设计用于手动和自动操作，并且与标准液体处理机器人平台兼容。这种兼容性简化了其融入高通量PCR工作流程的过程。此外，我们还开发了支持其自动化功能的软件。我们的结果表明，经过特殊设计的96孔板和HTGR能够有效地挤压真菌孢子，以100%的效率释放出足够用于PCR筛选的基因组DNA。这种基因组释放器能够在数分钟内从96个样本中制备好可用于PCR的基因组DNA，无需提取缓冲液。它对多种微生物和细胞类型的适应性使其成为一种多功能工具，可显著推动生物制造过程。
