Quantification of Jκ signal end breaks in developing B cells by blunt-end linker ligation and qPCR》.pdf

Journal of Immunological Methods 296 (2005) 19– 30 /locate/jim Research paper Quantification of Jn signal end breaks in developing B cells by blunt-end linker ligation and qPCR John D. Curry, Lydia Li, Mark S. Schlissel* University of California at Berkeley, Division of Immunology, Molecular and Cellular Biology, 439 Life Sciences Addition, Berkeley, CA 94720-3200, United States Received 9 September 2004; received in revised form 14 October 2004; accepted 18 October 2004 Available online 23 November 2004 Abstract Introduction of a double-strand DNA break at the junction between a rearranging gene segment and its flanking recombination signal sequence (RSS) is the first step of V(D)J recombination. Such DNA breaks can be detected by either Southern blot hybridization or ligation-mediated PCR. While Southern blotting is easily quantifiable, it is often insufficiently sensitive and while LM-PCR is far more sensitive, it is poorly quantifiable. Reported here is a LM-qPCR assay which relies on real-time qPCR to provide an absolute measure of recombinase-mediated, or any other specific, double-strand DNA break in genomic DNA. The efficiency of the initial ligation reaction was found to be relatively low with just 3% of potential targets undergoing linker ligation. Using this assay, approximately 16% of murine bone marrow pre-B cells were determined to contain a dsDNA break adjacent to the Jn1 gene segment. In addition, the kinetics of Jn1 dsDNA breaks in a temperature-sensitive cell line induced to recombine its n locus was