Bhuiyan Afsana, Asakawa Shuichi
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Front Bioeng Biotechnol. 2023 Mar 9;11:1115159. doi: 10.3389/fbioe.2023.1115159. eCollection 2023.
Non-coding repeat expansion causes several neurodegenerative diseases, such as fragile X syndrome, amyotrophic lateral sclerosis/frontotemporal dementia, and spinocerebellar ataxia (SCA31). Such repetitive sequences must be investigated to understand disease mechanisms and prevent them, using novel approaches. However, synthesizing repeat sequences from synthetic oligonucleotides is challenging as they are unstable, lack unique sequences, and exhibit propensity to make secondary structures. Synthesizing long repeat sequence using polymerase chain reaction is often difficult due to lack of unique sequence. Here, we employed a rolling circle amplification technique to obtain seamless long repeat sequences using tiny synthetic single-stranded circular DNA as template. We obtained 2.5-3 kbp uninterrupted TGGAA repeats, which is observed in SCA31, and confirmed it using restriction digestion, Sanger and Nanopore sequencing. This cell-free, cloning method may be applicable for other repeat expansion diseases and be used to produce animal and cell culture models to study repeat expansion diseases and .
非编码重复序列扩增会引发多种神经退行性疾病，如脆性X综合征、肌萎缩侧索硬化症/额颞叶痴呆以及脊髓小脑共济失调（SCA31）。必须采用新方法对这类重复序列进行研究，以了解疾病机制并预防疾病。然而，从合成寡核苷酸合成重复序列具有挑战性，因为它们不稳定，缺乏独特序列，并且易于形成二级结构。由于缺乏独特序列，使用聚合酶链反应合成长重复序列通常很困难。在这里，我们采用滚环扩增技术，以微小的合成单链环状DNA为模板，获得无缝长重复序列。我们获得了在SCA31中观察到的2.5 - 3kbp不间断的TGGAA重复序列，并通过限制性酶切、桑格测序和纳米孔测序对其进行了确认。这种无细胞克隆方法可能适用于其他重复序列扩增疾病，并可用于建立动物和细胞培养模型来研究重复序列扩增疾病。
