In vitro recombination of bacteriophage T7 DNA detected by a direct physical assay.
We developed a simple, direct, physical assay to detect genetic recombination of bacteriophage T7 DNA in vitro. In this assay two mature T7 DNA molecules, each having a unique restriction enzyme site, are incubated in the presence of a cell-free extract from T7-infected Escherichia coli cells. After extraction of the DNA, restriction enzyme digestion, and agarose gel electrophoresis, genetic recombination is detected by the appearance of a novel recombinant DNA band. Recombination frequencies as high as 13% have been observed. We used this assay to determine the genetic requirements for in vitro recombination. In agreement with results obtained previously with a biological assay, T7 recombination in vitro appears to proceed via two distinct pathways.
D004279:DNA, Viral / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D009154:Mutation; D009693:Nucleic Acid Hybridization; D011995:Recombination, Genetic; D013604:T-Phages / Q000235:genetics*
D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases; D005092:Exonucleases