Quantification of Jkappa signal end breaks in developing B cells by blunt-end linker ligation and qPCR.
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 Jkappa1 gene segment. In addition, the kinetics of Jkappa1 dsDNA breaks in a temperature-sensitive cell line induced to recombine its kappa locus was determined.
Curry
John D
JD
University of California at Berkeley, Division of Immunology, Molecular and Cellular Biology, 439 Life Sciences Addition, Berkeley, CA 94720-3200, USA.
Li
Lydia
L
Schlissel
Mark S
MS
D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry / Q000276:immunology*; D001854:Bone Marrow Cells / Q000737:chemistry / Q000276:immunology; D004247:DNA / Q000032:analysis / Q000235:genetics; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics; D015328:Gene Rearrangement, B-Lymphocyte, Light Chain; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D007133:Immunoglobulin Joining Region / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic / Q000235:genetics; D045567:VDJ Recombinases / Q000502:physiology
D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D009687:Nuclear Proteins; C497571:Rbpj protein, mouse; D004247:DNA; D045567:VDJ Recombinases