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ssDNA Tools

Single-stranded oligonucleotides may provide a new tool for homologous recombination and genome modification.

Jonathan Weitzman(jonathanweitzman@hotmail.com)

Homologous recombination of DNA is a powerful tool for chromosome engineering experiments, but is a rare event, requiring long stretches of homology and complex reactions. Phage-mediated systems allow efficient recombination of linear DNA with relatively short homologies. In the June 5 Proceedings of the National Academy of Sciences, Ellis et al. describe an efficient recombination system that uses short synthetic single-stranded DNA (ssDNA) (Proc Natl Acad Sci USA 2001, 98:6742-6746). They show that oligonucleotides as short as 30 nucleotides could be used to correct 'amber' mutations in the E. coligalK gene using the bacteriophage lambda Red system (with efficiencies up to 6%). Only the lambda Beta protein is absolutely required for ssDNA recombination. The authors propose a recombination mechanism in which the Beta protein binds and anneals the ssDNA to a complimentary single-strand near the DNA replication fork. They suggest that ssDNA may prove useful for chromosome...

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