Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation
Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation
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DOI：
10.1093/nar/gni129
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Larionov, V
中科院分区：
文献类型：
--
作者：
Ebersole, T;Okamoto, Y;Larionov, V
人类人工染色体（HACs）为研究动粒的形成和开发具有基因治疗潜力的新一代载体提供了一个独特的机会。研究HACs中着丝粒串联重复序列的结构和功能关系需要有效地操纵重复序列亚结构的能力。我们在这里描述了一种新的方法，快速扩增人类α串联重复的几百个碱基对到长DNA阵列高达120 kb。该方法包括体外重复序列的滚环扩增（RCA）和通过酵母中的体内重组组装RCA产物。当转化到人细胞中时，合成阵列在HAC形成中是胜任的。由于短的多聚体在扩增前可以很容易地被修饰，这种新技术可以识别对动粒接种至关重要的重复单体区域。该方法可能有更广泛的应用，在阐明其他串联重复在染色体组织和动力学的作用。
Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.