DNA cloning by homologous recombination in Escherichia coli.
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.
Zhang
Y
Y
Gene Expression Program, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Muyrers
J P
JP
Testa
G
G
Stewart
A F
AF
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D005090:Exodeoxyribonucleases