期刊
ANALYTICAL BIOCHEMISTRY
卷 479, 期 -, 页码 48-50出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2015.03.025
关键词
DNA repair; Resection; Single-stranded DNA; Double-strand break repair
资金
- Cancer Prevention and Research Institute of Texas, United States [RP110465-P4]
Single-stranded DNA (ssDNA) intermediates are formed in multiple cellular processes, including DNA replication and recombination. Here, we describe a quantitative polymerase chain reaction (qPCR)-based assay to quantitate ssDNA intermediates, specifically the 3' ssDNA product of resection at specific DNA double-strand breaks induced by the AsiSI restriction enzyme in human cells. We protect the large mammalian genome from shearing by embedding the cells in low-gelling-point agar during genomic DNA extraction and measure the levels of ssDNA intermediates by qPCR following restriction enzyme digestion. This assay is more quantitative and precise compared with existing immunofluorescence-based methods. (C) 2015 Elsevier Inc. All rights reserved.
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