Wang Jiancai, Xu Ronghua, Liu Aizhong
School of Life Sciences, University of Science and Technology of China, Hefei, People's Republic of China; Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, People's Republic of China.
Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, People's Republic of China.
PLoS One. 2014 Sep 22;9(9):e107907. doi: 10.1371/journal.pone.0107907. eCollection 2014.
Rapid and efficient construction of expression vectors and subsequent transformation are basic recombinant methods for the investigation of gene functionality. Although novel cloning methods have recently been developed, many laboratories worldwide continue to use traditional restriction digestion-ligation methods to construct expression vectors owing to financial constraints and the unavailability of appropriate vectors. We describe an improved restriction digestion-ligation (IRDL) cloning method that combines the advantage of directional cloning from double digestion-ligation with that of a low background observed by using a positive selection marker gene ccdB to facilitate digestion and ligation in a single tube. The IRDL cloning overcomes the time-consuming and laborious limits of traditional methods, thereby providing an easy-to-use, low-cost, and one-step strategy for directional cloning of target DNA fragments into an expression vector. As a proof-of-concept example, we developed two yeast vectors to demonstrate the feasibility and the flexibility of the IRDL cloning method. This method would provide an effective and easy-to-use system for gene cloning and functional genomics studies.
快速高效地构建表达载体以及随后的转化是研究基因功能的基本重组方法。尽管最近开发了新的克隆方法，但由于资金限制和缺乏合适的载体，全球许多实验室仍继续使用传统的限制性酶切连接方法来构建表达载体。我们描述了一种改进的限制性酶切连接（IRDL）克隆方法，该方法将双酶切连接的定向克隆优势与使用阳性选择标记基因ccdB观察到的低背景优势相结合，以便在单个试管中促进酶切和连接。IRDL克隆克服了传统方法耗时费力的局限性，从而为将目标DNA片段定向克隆到表达载体中提供了一种易于使用、低成本的一步策略。作为概念验证示例，我们开发了两种酵母载体以证明IRDL克隆方法的可行性和灵活性。该方法将为基因克隆和功能基因组学研究提供一个有效且易于使用的系统。
