Gene Cloning Methods Without Ligase Enzyme

Document Type : Research Paper

Authors

1 Assistant Professor, Department of Biotechnology, Faculty of Agriculture, Bu-Ali sina University, Hamedan

2 Assistant Professor, Department of Agronomy and Plant breeding, Faculty of Agriculture, Bu-Ali sina University, Hamedan

Abstract

The cloning of a DNA fragment into a plasmid vector is a procedure in recombinant DNA technology. The most common methods for cloning require the use of DNA ligase and restriction enzymes which are less efficient. Different Ligation-free  cloning methods have been developed in which of them, Gateway and USER friendly technologies  have been commercialized. Gateway technology is a rapid and efficient method of cloning base on bacteriophage lambda site-specific recombination system. The components of the lambda recombination system are modified to improve the efficiency of the system. Recombination occurs between site-specific attachments. In this technology, a target fragment is cloned into a entry vector. Transfer of DNA from entry vector to other vectors easily can be done without any restriction enzymes and ligase in one hour recombination reaction.  In the USER (uracil-specific excision reagent) Friendly Cloning, vector-specific PCR primers which contain one uracil per primer are designed and the target DNA is amplified with Taq DNA polymerase. The resulting PCR products are treated with the special enzyme to create unique 3´ single-stranded extensions which can then anneal to the supplied linearized vector without ligation enzyme. By varying the design of the PCR primers, the protocol is easily adapted to perform DNA manipulations. In this review key aspects and advantages of these methods are discussed.

Keywords

Volume 3, Issue 1 - Serial Number 1
November 2012
Pages 51-61

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