Abstract - Deletion of DNA sequences of using a polymerase chain reaction based approach

Deletion of DNA sequences of using a polymerase chain reaction based approach

Pablo Pérez-Pinera
Departamento de Morfología y Biología Celular
Facultad de Medicina
C/. Julián Clavería s/n, CP:33006
Oviedo, Spain
Tel: 34 985103614
Fax: 34 985103618
E-mail: pab_correo@yahoo.com

Manuel Menéndez-González*
Servicio de Neurología
Hospital Álvarez-Buylla
Murias s/n, CP: 33616
Mieres, Spain
Tel: 34 985458500
Fax: 34 985458500
E-mail: manuelmenendez@gmail.com

José Antonio Vega
Departamento de Morfología y Biología Celular
Facultad de Medicina
C/. Julián Clavería s/n, CP:33006
Oviedo, Spain
Tel: 34 985103614
Fax: 34 985103618
E-mail: javega@correo.uniovi.es

*Corresponding author

Keywords: deletion, mutagenesis, polymerase chain reaction, plasmid.

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.