Figure 1.

Identification of DNA breaks. DNA is denatured in the presence of a 5′-biotinylated (BBBB) primer specific for the gene of interest and the primer is extended with Sequenase. Synthesis terminates at the 5′-end of the template to produce a duplex with a blunt end. This duplex is ligated to a linker, and biotinylated DNAs are recovered by binding to streptavidin-coated magnetic beads. Following dilution, the extension products are amplified by nested PCR and breaksites identified by batch sequencing. See Materials and Methods for details.