The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination, for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.

DNA cloning by homologous recombination in Escherichia coli / Y. Zhang, J.P.P. Muyrers, G. Testa, A.F. Stewart. - In: NATURE BIOTECHNOLOGY. - ISSN 1087-0156. - 18:12(2000), pp. 1314-1317. [10.1038/82449]

DNA cloning by homologous recombination in Escherichia coli

Testa G.;
2000

Abstract

The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination, for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.
Artificial chromosomes; bacteriophage-lambda; replacement
Settore BIO/11 - Biologia Molecolare
Settore BIO/13 - Biologia Applicata
Settore BIO/10 - Biochimica
Settore BIO/18 - Genetica
Settore MED/03 - Genetica Medica
Article (author)
File in questo prodotto:
File Dimensione Formato  
Zhang Nat Biotech 2000.pdf

non disponibili

Tipologia: Publisher's version/PDF
Dimensione 95.53 kB
Formato Adobe PDF
95.53 kB Adobe PDF   Visualizza/Apri   Richiedi una copia
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/802307
Citazioni
  • ???jsp.display-item.citation.pmc??? 149
  • Scopus 336
  • ???jsp.display-item.citation.isi??? 305
social impact