Recombineering

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Recombineering (English from recombi nogenic engineering ) describes a molecular biological method for manipulating DNA , based on the homologous recombination of Escherichia coli , which is known by the synonym Red / ET recombination .

Recombineering is based on the homologous recombination mediated by proteins derived from bacteriophages in Escherichia coli . Two homologous systems are known. The RecE / RecT the Rac prophage and the red - Operon consisting of Redγ, Redβ and Redα from λ bacteriophage . Both systems allow the efficient and exact exchange of freely selectable DNA segments between two different DNA molecules. The red recombination system is used most frequently. The exchange of DNA takes place via two homologous (similar or identical) regions flanking the target fragment with a length of 30 to 50 bp.

advantages

Recombineering can be viewed as a further development of conventional cloning , which works independently of restriction enzymes through the use of homologous recombination . Therefore, this method is particularly suitable for manipulating large vectors (> 100 kBp ) such as. B. Bacterial Artificial Chromosomes (BACs). Recombineering allows the desired modifications without leaving additional DNA fragments.

literature

  • Zhang et al .: A new logic for DNA engineering using recombination in Escherichia coli. In: Nat Genet . (1998) vol. 20 (2) pp. 123-8
  • Muyrers et al .: Rapid modification of bacterial artificial chromosomes by ET-recombination. In: Nucleic Acids Res. (1999) vol. 27 (6) pp. 1555-7
  • Ellis et al .: High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. In: Proc Natl Acad Sci USA . (2001) vol. 98 (12) pp. 6742-6
  • Muyrers et al .: Techniques: Recombinogenic engineering - new options for cloning and manipulating DNA. In: Trends Biochem Sci . (2001) vol. 26 (5) pp. 325-31
  • Copeland et al .: Recombineering: a powerful new tool for mouse functional genomics. In: Nat Rev Genet . (2001) vol. 2 (10) pp. 769-79
  • Court et al .: Genetic engineering using homologous recombination. In: Annu Rev Genet . (2002) vol. 36 pp. 361-88