Centre for Organismal Studies Heidelberg, Heidelberg University, Heidelberg, Germany.
Heidelberg Biosciences International Graduate School, Heidelberg University, Heidelberg, Germany.
PLoS One. 2019 Mar 5;14(3):e0213317. doi: 10.1371/journal.pone.0213317. eCollection 2019.
In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, the handling-time and material costs per sample remain major limiting factors. Here we present a robust method for low-cost DIY-pipet tips addressing these needs; i.e. using a cellulose filter disc inserted into a regular pipet tip. These filter-in-tips allow for a rapid, stand-alone four-step genotyping workflow by simply binding the DNA contained in the primary lysate to the cellulose filter, washing it in water and eluting it directly into the buffer for the downstream application (e.g. PCR). This drastically cuts down processing time to maximum 30 seconds per sample, with the potential for parallelizing and automation. We show the ease and sensitivity of our procedure by genotyping genetically modified medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos (targeted by CRISPR/Cas9 knock-out and knock-in) in a 96-well plate format. The robust isolation and detection of multiple alleles of various abundancies in a mosaic genetic background allows phenotype-genotype correlation already in the injected generation, demonstrating the reliability and sensitivity of the protocol. Our method is applicable across kingdoms to samples ranging from cells to tissues i. e. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips.
在 CRISPR 基因编辑和基因筛选的时代，人们对快速可靠的核酸提取管道的需求越来越大，以便对大型和多样化的样本集进行快速基因分型。尽管当前的工作流程不断改进，但每个样本的处理时间和材料成本仍然是主要的限制因素。在这里，我们提出了一种针对低成本 DIY 移液管吸头的稳健方法，以满足这些需求；即将纤维素滤膜插入常规移液管吸头中。这些滤膜吸头可通过将初级裂解物中包含的 DNA 直接绑定到纤维素滤膜上，在水中洗涤，然后直接将其洗脱到下游应用（例如 PCR）的缓冲液中，从而实现快速、独立的四步基因分型工作流程。这将处理时间缩短至每个样本最长 30 秒，并且具有并行化和自动化的潜力。我们通过以 96 孔板格式对基因修饰的斑马鱼（Danio rerio）和日本青鳉（Oryzias latipes）胚胎（通过 CRISPR/Cas9 敲除和敲入靶向）进行基因分型，展示了我们方法的简便性和灵敏度。在镶嵌遗传背景下，我们能够稳健地分离和检测各种丰度的多个等位基因，从而在注射一代中就可以进行表型与基因型的相关性分析，证明了该方案的可靠性和灵敏度。我们的方法适用于从细胞到组织的各种样本，包括植物幼苗、成年苍蝇、小鼠细胞培养物和组织以及成年鱼鳍。
