Data/hora: 04/06/2024 03:00:00

Provedor de dados: | 69
País: | Chile
Título: | Deletion of DNA sequences of using a polymerase chain reaction based approach
Autores: | Pérez-Pinera,Pablo; Menéndez-González,Manuel; Vega,José Antonio.
Data: | 2006-10-01
Ano: | 2006
Palavras-chave: | Deletion; Mutagenesis; Polymerase chain reaction; Plasmid.
Resumo: | 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.
Tipo: | Journal article
Idioma: | Inglês
Identificador: | http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582006000500018
Editor: | Pontificia Universidad Católica de Valparaíso
Formato: | text/html
Fonte: | Electronic Journal of Biotechnology  v.9 n.5 2006