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.
Highlights
Molecular cloning is an essential tool in modern molecular biology and biotechnology
Conventional cloning methods based on restriction digestion and ligation have played a critical role in the construction of recombinant DNA molecules
Because of its laborious and time-consuming nature, as well as limitations in restriction sites in the multiple cloning sites (MCS), a wide variety of novel cloning technologies have been developed as alternate methodologies to restriction digestion and ligation
Summary
Molecular cloning is an essential tool in modern molecular biology and biotechnology. The cloning of an insert into an expression vector involves the use of Type II restriction enzymes to generate the appropriate DNA fragments encompassing the target sequence, followed by modification of DNA ends to generate blunt or sticky ends, and ligation of the DNA fragments to generate plasmids or other types of DNA vectors [1] This procedure is extremely laborious and time consuming applied to a high-throughput format and is often limited by the relatively few restriction sites available [2]. The LIC and SLIC methods use the exonuclease activity of T4 DNA polymerase to generate long cohesive ends between the vector and the target DNA fragments for subsequent annealing and in vivo repair These methods require multiple experimental steps and long primers, which usually increase the cost and difficulty of PCR amplification. Since this system is based on the action of the type II restriction enzyme BsaI, the backbone expression vectors require removal of internal BsaI sites [7]
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