Trends in Biochemical Sciences
ReviewTechniques: Recombinogenic engineering–new options for cloning and manipulating DNA
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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