Research Notes

Researchers at the National Cancer Institute in Frederick, Md. have developed a new tool for editing and repairing bacterial DNA in vivo using *-mediated homologous recombination. Originally used with chromosomal genes in yeast and Escherichia coli, this technique soon could be used with genes cloned on plasmids, which would allow scientists to study other pathogens and correct mutations or create markers in eukaryotic cells. A team led by Donald Court, head of the molecular control and genetics section at NCI, introduced synthetic, single-stranded oligonucleotides, some as short as 30 base pairs, into E. coli DNA using Beta protein of phage * (H. Ellis et al., "High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides," Proceedings of the National Academy of Sciences, 98:6742-6, June 5, 2001). This protein binds the ssDNA donor fragment to a complementary single strand near the replication fork; DNA polymerase and ligase then join this fragment to the chromosome to create the recombinant. The scientists noted more recombination efficiency in the lagging strand, probably due to the increase in single-strand regions during lagging strand synthesis. "The main idea is that we're using ssDNA instead of dsDNA," explains Court. "This method facilitates creating or repairing mutations in a genome with ssDNA donors." Eventually, this "recombineering," as it has been called, might replace traditional DNA repair using restriction enzymes and DNA ligase, but only in certain areas, according to Court. "It's so precise. We can create the exact kinds of clones we want, right now in E. coli, but eventually in mouse or even human cells. Some of our collaborators are working on that now."