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Directed PCR-free engineering of highly repetitive DNA sequences

机译：无需PCR的高度重复DNA序列工程

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Background Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. Results For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6- SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro . The His6- SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays. Conclusion Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.
机译：背景技术在自然界中通常发现高度重复的核苷酸序列，例如。在端粒，微卫星DNA，真核生物信使RNA的聚腺嘌呤（poly（A））尾巴以及与基因组中三核苷酸重复扩增有关的几种遗传性人类疾病中。因此，研究重复序列具有生物学，生物技术和医学意义。然而，克隆此类重复性DNA序列是具有挑战性的，因为缺乏独特的引物结合位点会阻碍基于特异性PCR的扩增，从而导致产生非特异性产物。结果为了免PCR产生重复的DNA序列，我们使用了反平行的寡核苷酸，其侧翼是IIS型核酸内切酶的限制性酶切位点。识别位点的排列允许重复序列的逐步和无缝延伸。这促进了重复DNA片段和编码具有任何所需长度的周期性氨基酸序列的多肽的开放阅读框的组装。通过这种策略，我们克隆了一系列的聚谷氨酰胺编码序列以及高度重复的聚腺嘌呤片段。这样的重复序列可以用于多种生物技术应用。例如，聚谷氨酰胺序列在大肠杆菌细胞中表达为His 6 -SUMO融合蛋白，以研究其体外聚集行为。 His 6 -SUMO部分可实现聚谷氨酰胺蛋白的亲和纯化，增加其溶解度，并控制聚合过程。我们成功地纯化了融合蛋白，并为它们在过滤器阻滞测定中的适用性提供了一个实例。结论我们的无缝克隆策略无需PCR，可通过简单的标准克隆程序直接有效地生成定义长度的高度重复性DNA序列。

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