Nucleic Acids Research, 2001, Vol. 29, No. 6 e33
© 2001 Oxford University Press
Breaksite batch mapping, a rapid method for assay and identification of DNA breaksites in mammalian cells
1Department of Molecular Biophysics and Biochemistry and 2Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8024, USA
DNA breaks occur during many processes in mammalian cells, including recombination, repair, mutagenesis and apoptosis. Here we report a simple and rapid method for assaying DNA breaks and identifying DNA breaksites. Breaksites are first tagged and amplified by ligation-mediated PCR (LM-PCR), using nested PCR primers to increase the specificity and sensitivity of amplification. Breaksites are then mapped by batch sequencing LM-PCR products. This allows easy identification of multiple breaksites per reaction without tedious fractionation of PCR products by gel electrophoresis or cloning. Breaksite batch mapping requires little starting material and can be used to identify either single- or double-strand breaks.
* To whom correspondence should be addressed at present address: Division of Neuropathology, Room 405, Department of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, USA. Tel: +1 216 368 1756; Fax: +1 216 368 2546; Email: qxk2@po.cwru.edu Correspondence may also be addressed to Nancy Maizels at present address: Departments of Immunology and Biochemistry, University of Washington Medical School, Room H474A HSB, 1959 NE Pacific Street, Box 357650, Seattle, WA 98195-7650, USA. Tel: +1 206 221 6876; Fax: +1 206 221 6781; Email: maizels@u.washington.edu