title: IRDL cloning: a one-tube, zero-background, easy-to-use, directional cloning method improves throughput in recombinant DNA preparation.
abstract: 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.
authors.affiliation: 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.
authors.forename: Jiancai
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, People's Republic of China.
authors.forename: Ronghua
authors.initials: R
authors.lastname: Xu
authors.affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, People's Republic of China; Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Kunming, People's Republic of China.
authors.forename: Aizhong
authors.initials: A
authors.lastname: Liu
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant / Q000235:genetics*; D005819:Genetic Markers; D005822:Genetic Vectors
chemical_list: D004274:DNA, Recombinant; D005819:Genetic Markers