Approximately 3% of the human genome is composed of short tandem repeat (STR) DNA sequence known as microsatellites, which can be found in both coding and non-coding regions. When associated with genic regions, expansion of microsatellite repeats beyond a critical threshold causes dozens of neurological repeat expansion disorders. To better understand the molecular pathology of repeat expansion disorders, precise cloning of microsatellite repeat sequence and expansion size is highly valuable. Unfortunately, cloning repeat expansions is often challenging and presents a significant bottleneck to practical investigation. Here, we describe a clear method for seamless and systematic cloning of practically any microsatellite repeat expansion. We use cloning and expansion of GGGGCC repeats, which are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as an example. We employ a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors. We describe methods to validate repeat expansion cloning, including diagnostic restriction digestion, PCR across the repeat, and next-generation long-read MinION nanopore sequencing. Validated cloning of microsatellite repeats beyond the critical expansion threshold can facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level.

大约 3% 的人类基因组由称为微卫星的短串联重复 (STR) DNA 序列组成，可在编码区和非编码区找到。当与基因区域相关时，微卫星重复序列的扩展超出临界阈值会导致数十种神经系统重复序列扩展障碍。为了更好地了解重复扩增障碍的分子病理学，精确克隆微卫星重复序列和扩增大小非常有价值。不幸的是，克隆重复扩展通常具有挑战性，并且是实际研究的重大瓶颈。在这里，我们描述了一种清晰的方法，用于几乎任何微卫星重复扩展的无缝和系统克隆。我们使用 GGGGCC 重复的克隆和扩展，例如，它们是肌萎缩侧索硬化 (ALS) 和额颞叶痴呆 (FTD) 的主要遗传原因。我们采用递归定向连接 (RDL) 技术来构建多个包含 GGGGCC 重复的载体。我们描述了验证重复扩增克隆的方法，包括诊断性限制性消化、重复 PCR 和下一代长读取 MinION 纳米孔测序。超过临界扩展阈值的微卫星重复的验证克隆可以促进在细胞和分子水平上逐步表征疾病机制。包括诊断性限制性消化、重复 PCR 和下一代长读长 MinION 纳米孔测序。超过临界扩展阈值的微卫星重复的验证克隆可以促进在细胞和分子水平上逐步表征疾病机制。包括诊断性限制性消化、重复 PCR 和下一代长读长 MinION 纳米孔测序。超过临界扩展阈值的微卫星重复的验证克隆可以促进在细胞和分子水平上逐步表征疾病机制。