Development of the SapI/AarI Incision Mediated Plasmid Editing Method.

Autor: Gasparyan HJ; Biology Department, Loyola Marymount University, 1 Loyola Maryount Dr., Los Angeles, CA 90045, United States., Kroh J; Biochemistry Program, Ohio State University, 1461 Mount Vernon Avenue, Marion, OH 43302, United States., Michael WM; Molecular and Computational Biology Section, Department of Biological Sciences, University of Southern California, 1050 Childs Way, Los Angeles, CA 90089, United States., Petreaca RC; Department of Molecular Genetics, Ohio State University, 1461 Mount Vernon Avenue, Marion, OH 43302, United States. Electronic address: petreaca.1@osu.edu.
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 2018 May 11; Vol. 430 (10), pp. 1426-1430. Date of Electronic Publication: 2018 Apr 05.
DOI: 10.1016/j.jmb.2018.03.030
Abstrakt: Plasmid engineering and molecular cloning is a virtually ubiquitous tool in biology. Although various methods have been developed for ligating DNA molecules or targeted mutagenesis of plasmids, each has its limitations. Many of the commonly used laboratory strategies are inefficient, while commercially available kits are quite costly and often specialized for highly specific circumstances. Here, we describe the SapI/AarI incision mediated plasmid editing (SIMPLE) method, which allows users to perform site-directed mutagenesis, deletions, and even short insertions into any plasmid in a single PCR reaction, using just one restriction enzyme. In addition, the SIMPLE method can be adapted to insert any sized DNA fragment into a vector using a two-step PCR approach, and can be used to ligate any number of DNA fragments with non-compatible ends in the specific order desired. The SIMPLE method provides researches an efficient and powerful tool with a broad range of applications for molecular cloning.
(Published by Elsevier Ltd.)
Databáze: MEDLINE
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