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 determined. D 2004 Elsevier B.V. All rights reserved. Keywords: qPCR; Blunt-ended ligation; dsDNA breaks; J kappa 1; Murine; Pre-B cells 1. Introduction Ligation of linkers to DNA molecules is a widely utilized technique in molecular biology. When com- bined with PCR, the ligation of a linker sequence to fragmented genomic DNA allows for the amplifica- tion of DNA sequences adjacent to a DNA break. First developed for genomic sequencing and in vivo DNA foot-printing (Mueller and Wold, 1989), ligation- mediated PCR (LM-PCR) assays have been adapted to detect DNA breaks introduced by the V(D)J recombinase (Schlissel et al., 1993; Roth et al., 1993). V(D)J recombination is a site-specific DNA recombination reaction essential for the generation of immune receptor diversity (reviewed in Jung and Alt, 2004; Gellert, 2002). Germline-encoded V, D, and J gene segments are rearranged to generate exons 0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2004.10.003 Abbreviations: qPCR, quantitative PCR; RSS, recombination signal sequence; LM, ligation-mediated; dsDNA, double-stranded DNA; SE, signal ends. * Corresponding author. Tel.: +1 510 643 2462; fax: +1 510 642 6845. E-mail address: mss@berkeley.edu (M.S. Schlissel). Journal of Immunological Methods 296 (2005) 19 – 30 www.elsevier.com/locate/jim