Stevenson Julian, Krycer James R, Phan Lisa, Brown Andrew J
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales, Australia.
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales, Australia ; Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
PLoS One. 2013 Dec 23;8(12):e83888. doi: 10.1371/journal.pone.0083888. eCollection 2013.
The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.
特定DNA序列的精确组装是分子生物学中的一项关键技术。传统的克隆技术在体外使用限制性内切酶和DNA连接，这可能会因缺乏合适的限制性酶切位点和低效的酶促步骤而受到阻碍。已经开发了许多不依赖连接的克隆技术，包括聚合酶不完全引物延伸（PIPE）克隆、序列和连接不依赖克隆（SLIC）以及重叠延伸克隆（OEC）。这些策略依赖于DNA聚合酶产生互补的突出端，无需特定的限制性酶切位点或连接，并能在短时间内以低成本实现高效率。在这里，我们概述并优化了这些技术，并确定了指导克隆项目设计的重要因素，包括避免PCR假象，如引物二聚体和载体质粒背景。实验使用了一个常见的报告载体和一组模块化引物来克隆大小不断增加的DNA片段。总体而言，PIPE只需很少的操作就能达到约95%的克隆效率，而SLIC能产生更多的转化子，但需要额外的步骤。我们的数据表明，对于小插入片段（<1.5 kb），OEC是一个不错的选择，只需要两个新引物，但对于较大的插入片段效果不佳。这些不依赖连接的克隆方法是研究人员分子工具包的重要组成部分。
