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How Red®/ET® Works

A superior technique for DNA manipulation, called Red/ET Recombination, was developed in A. Francis Stewart's Laboratory at EMBL. This technical breakthrough is precise and independent of the presence of restriction sites and the size of the DNA molecule to be modified. Therefore, problems previously incurred by traditional DNA engineering techniques [1,2] have come to an end [3, 4]. Furthermore this innovative technology is significantly less laborious than traditional DNA manipulation techniques and therefore has large savings on time and cost [5].

In Red/ET Recombination, also referred to as λ-mediated recombination, target DNA molecules are precisely altered by homologous recombination in strains of E.coli which express phage-derived protein pairs, either RecE/RecT from the Rac prophage, or Redα/Redβ from λ phage [6]. These protein pairs are functionally and operationally equivalent. RecE and Redα are 5->3 exonucleases, and RecT and Redβ are DNA annealing proteins. A functional interaction between RecE and RecT, or between Redα and Redβ is also required in order to catalyse the homologous recombination reaction.

Recombination

Fig.1 Mechanism of Red/ET Recombination

Recombination occurs through homology regions, which are stretches of DNA shared by the two molecules that recombine. Since the sequence of the homology regions can be chosen freely, any position on a target molecule can be specifically altered. Homologous recombination allows the exchange of genetic information between two DNA molecules in a precise, specific and faithful manner, qualities that are optimal for DNA engineering regardless of size.

Red/ET Recombination

Fig.2 Red/ET Recombination
The central step in Red/ET Recombination is the crossover step between a targeting construct containing homology arms (hm) and the target which can be a gene locus on the E.coli chromosome or any other stretch of DNA in a BAC or plasmid vector.

One of the biggest advantages of Red/ET Recombination is that it is not just limited to its unique ability to clone and subclone large DNA molecules but also it presents a variety of new ways to simplify conventional DNA engineering exercises [5, 7]. Red/ET Recombination allows every type of DNA modification possible. It becomes trivial to direct changes to a chosen DNA sequence or to introduce point mutations at any chosen site of a target DNA molecule regardless of its size.

Applications of Red/ET

Fig.3 Applications of Red/ET
Red/ET allows every type of DNA engineering possible regardless of target size or type of modification.

The potential to use homologous recombination for DNA engineering had long been recognised, and methods which harnessed the endogenous homologous recombination system (termed RecA-dependent recombination) in E.coli, the premier cloning host, were developed [6]. However this endogenous system has many limitations. It is impossible to use linear DNA molecules as they become rapidly degraded. Furthermore, recombination of circular molecules require relatively long homology regions and the ratio of intended to unwanted recombination products is extremely low. None of these limitations are encountered with Red/ET Recombination.

References

[1] Cohen S.N, Chang A.C., Boyer, H.W, and Heiling , R.B. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA 70 (1973) 3240-3244

[2] Mullis K ,Faloona F, Scharf S, Saiki R, Horn, G, Erlich, H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction 51 (1986) Cold Spring Harb. Symp. Quant. Biol. 263-273

[3] Zhang Y, Buchholz F, Muyrers J.P.P. and Stewart A.F. A new logic for DNA engineering using recombination in E.Coli. Nature Genetics 20 (1998) 123-128

[4] Zhang Y, Muyrers J.P.P., Testa G and Stewart A.F. DNA cloning by homologous recombination in E.Coli. Nature Biotechnology 18 (2000) 1314-1317

[5] Muyrers, J.P.P., Zhang, Y., Testa, G., Stewart, A.F. Rapid modification of bacterial artificial chromosomes by ET-recombination Nucleic Acids Res. 27, 1555-1557 (1999).

[6] Muyrers, J.P.P., Zhang, Y., Stewart, A.F. ET cloning: Think recombination first. Genetic Engineering, Principles and Methods (Ed. J.K. Setlow), 22, 77-98 Kluwer Academic/Plenum Publishers, NY. (2000).

[7] Muyrers J.P.P, Zhang Y, and Stewart A.F. TIBS: Recombinogenic engineering- new options for cloning and manipulating DNA 26 (2001)325-331


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