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

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 r...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Pérez-Pinera,Pablo, Menéndez-González,Manuel, Vega,José Antonio
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2006
Materias:	deletion mutagenesis polymerase chain reaction plasmid
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582006000500018
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:scielo:S0717-34582006000500018
record_format	dspace
spelling	oai:scielo:S0717-345820060005000182007-01-04Deletion of DNA sequences of using a polymerase chain reaction based approachPérez-Pinera,PabloMenéndez-González,ManuelVega,José Antonio 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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.9 n.5 20062006-10-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582006000500018en
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	deletion mutagenesis polymerase chain reaction plasmid
spellingShingle	deletion mutagenesis polymerase chain reaction plasmid Pérez-Pinera,Pablo Menéndez-González,Manuel Vega,José Antonio Deletion of DNA sequences of using a polymerase chain reaction based approach
description	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.
author	Pérez-Pinera,Pablo Menéndez-González,Manuel Vega,José Antonio
author_facet	Pérez-Pinera,Pablo Menéndez-González,Manuel Vega,José Antonio
author_sort	Pérez-Pinera,Pablo
title	Deletion of DNA sequences of using a polymerase chain reaction based approach
title_short	Deletion of DNA sequences of using a polymerase chain reaction based approach
title_full	Deletion of DNA sequences of using a polymerase chain reaction based approach
title_fullStr	Deletion of DNA sequences of using a polymerase chain reaction based approach
title_full_unstemmed	Deletion of DNA sequences of using a polymerase chain reaction based approach
title_sort	deletion of dna sequences of using a polymerase chain reaction based approach
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2006
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582006000500018
work_keys_str_mv	AT perezpinerapablo deletionofdnasequencesofusingapolymerasechainreactionbasedapproach AT menendezgonzalezmanuel deletionofdnasequencesofusingapolymerasechainreactionbasedapproach AT vegajoseantonio deletionofdnasequencesofusingapolymerasechainreactionbasedapproach
_version_	1718441760321961984

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

Ejemplares similares