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重复DNA序列的克隆与扩增。

Cloning and expansion of repetitive DNA sequences.

作者信息

Williams Sophie L, Coster Gideon

机构信息

Genome Replication lab, Division of Cancer Biology, Institute of Cancer Research, Chester Beatty Laboratories, London, United Kingdom.

Genome Replication lab, Division of Cancer Biology, Institute of Cancer Research, Chester Beatty Laboratories, London, United Kingdom.

出版信息

Methods Cell Biol. 2024;182:167-185. doi: 10.1016/bs.mcb.2022.10.013. Epub 2022 Nov 28.

Abstract

Repeat and structure-prone DNA sequences comprise a large proportion of the human genome. The instability of these sequences has been implicated in a range of diseases, including cancers and neurodegenerative disorders. However, the mechanism of pathogenicity is poorly understood. As such, further studies on repetitive DNA are required. Cloning and maintaining repeat-containing substrates is challenging due to their inherent ability to form non-B DNA secondary structures which are refractory to DNA polymerases and prone to undergo rearrangements. Here, we describe an approach to clone and expand tandem-repeat DNA without interruptions, thereby allowing for its manipulation and subsequent investigation.

摘要

重复且易于形成特定结构的DNA序列在人类基因组中占很大比例。这些序列的不稳定性与一系列疾病有关,包括癌症和神经退行性疾病。然而,其致病机制尚不清楚。因此,需要对重复DNA进行进一步研究。由于含重复序列的底物具有形成非B型DNA二级结构的固有能力,这种结构难以被DNA聚合酶识别且容易发生重排,因此克隆和维持含重复序列的底物具有挑战性。在这里,我们描述了一种不间断地克隆和扩增串联重复DNA的方法,从而便于对其进行操作和后续研究。

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