Quantification of JK signal end breaks in developing B cells by blunt-end linker ligation and qPCR

被引:5
作者
Curry, JD [1 ]
Li, L [1 ]
Schlissel, MS [1 ]
机构
[1] Univ Calif Berkeley, Div Immunol Mol & Cellular Biol, Berkeley, CA 94720 USA
关键词
qPCR; blunt-ended ligation; dsDNA breaks; J kappa 1; murine; pre-B cells;
D O I
10.1016/j.jim.2004.10.003
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 K locus was determined. (C) 2004 Elsevier B.V. All rights reserved.
引用
收藏
页码:19 / 30
页数:12
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