Review
Techniques: Recombinogenic engineering–new options for cloning and manipulating DNA

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Abstract

Driven by the needs of functional genomics, DNA engineering by homologous recombination in Escherichia coli has emerged as a major addition to existing technologies. Two alternative approaches, RecA-dependent engineering and ET recombination, allow a wide variety of DNA modifications, including some which are virtually impossible by conventional methods. These approaches do not rely on the presence of suitable restriction sites and can be used to insert, delete or substitute DNA sequences at any desired position on a target molecule. Furthermore, ET recombination can be used for direct subcloning and cloning of DNA sequences from complex mixtures, including bacterial artificial chromosomes and genomic DNA preparations. The strategies reviewed in this article are applicable to modification of DNA molecules of any size, including very large ones, and present powerful new avenues for DNA manipulation in general.

Section snippets

Homologous recombination and DNA engineering: recombinogenic engineering

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

Because homologous

Recombinogenic engineering in E. coli

Although E. coli is the premier host for conventional DNA engineering, Saccharomyces cerevisiae was, until recently, the preferred host for recombinogenic engineering. This situation was due to the remarkable proficiency of S. cerevisiae at homologous recombination, combined with certain complications inherent in the endogenous E. coli homologous recombination mechanism. Recombinogenic engineering in S. cerevisiae is straightforward because the linear DNA flanked by short homology regions can

RecA-dependent recombinogenic engineering

Consistent with the difficulties of using linearized DNA in E. coli, early findings with RecA-dependent recombinogenic engineering reported some successes using intermolecular recombination between two circular molecules 30, 31. A significant advance in RecA-dependent strategies was the inclusion of a temperature-sensitive (ts) plasmid origin. This permitted the construction of a targeting plasmid, grown at the permissive temperature, followed by a first round of homologous recombination to

Recombinogenic engineering using ET recombination

An alternative recombinogenic engineering strategy was developed whereby recombination is not dependent on RecA, but instead mediated by phage-derived protein pairs, either RecE/RecT from the Rac phage or Redα/Redβ, from λ phage 5, 6. To coin a simple term, this recombinogenic engineering strategy was initially termed ET recombination (orET cloning) 5 and has also been called λ-mediated recombination 33 and GET recombination 14. As established from both fundamental studies 22, 23, 36 and

Limiting the recombinogenic window

Recombinogenic engineering, either RecA-dependent or by ET recombination, occurs through homology regions. Any two regions homologous to each other within a recombinogenic host can recombine. In practical terms, this means that unintended additional homology regions in the targeting DNA should be avoided. Probable unintended homologies include all or parts of plasmid origins, selectable markers or other commonly used DNA sequences such as fragments carried over from common cloning vectors.

Recent developments

Following the recombinogenic engineering initiatives pioneered in S. cerevisiae 20, 21, the potential of ET recombination for direct cloning and subcloning was explored. The principle of this strategy is illustrated in Fig. 3. In this variation, the linear targeting molecule is a PCR-amplified plasmid backbone that contains a selectable gene and an origin of replication. The oligonucleotides used for PCR also contain homology regions that are chosen to define the exact boundaries of the DNA

Concluding remarks and perspectives

The strategies presented outline how precise modifications, including single-base changes, and variably sized insertions and deletions, can be made to DNA molecules in E. coli of any size. Recombinogenic engineering strategies will undoubtedly accelerate progress in functional genomics, as they allow straightforward engineering of large DNA molecules, which are important for functional studies of genes in all aspects such as development, homeostasis and disease. Importantly, the strategies also

Acknowledgements

Recombinogenic engineering in E. coli is built on 35 years of fundamental research, which could not be acknowledged here by reference. Please see Refs 22,23 as a starting point. We thank Inhua Muyrers-Chen, Michelle Meredyth, Vladimir Benes and the referees for comments on the manuscript. Our work in the laboratory was supported by grants from the Volkswagen Foundation, Program on Conditional Mutagenesis and the NIH, National Institute for Aging.

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