Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
Vol. 9, No. 5, 2006, pp. 604-609
Bioline Code: ej06082
Full paper language: English
Document type: Research Article
Document available free of charge
Electronic Journal of Biotechnology, Vol. 9, No. 5, 2006, pp. 604-609
© Copyright 2006 - Pontificia Universidad Católica de Valparaíso -- Chile
Deletion of DNA sequences of using a polymerase chain reaction based approach|
Pérez-Pinera, Pablo; Menéndez-González, Manuel & Vega, José Antonio
We developed a simple, rapid and reliable method to delete DNA fragments in plasmids using a polymerase chain reaction based amplification of the circular DNA sequence that excludes the fragment to be deleted. The primers are designed to contain a non-complementary 5' sequence consisting of a restriction enzyme target sequence. Following PCR amplification, the plasmid is digested with Dpn I to eliminate the template DNA, with the chosen restriction enzyme, and ligated. The only limitation is the selection of the restriction enzyme target sequence that must not be present in the original plasmid. The method is straightforward in its execution and success relies on a meticulous primer design that permits us obtain 100% of transformants containing the desired mutation. The extraordinary simplicity of the method makes it a valuable tool to generate DNA deletions in plasmids and to study the effects of those deletions in protein function.
deletion, mutagenesis, polymerase chain reaction, plasmid.
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