title: Direct measurement of single-stranded DNA intermediates in mammalian cells by quantitative polymerase chain reaction.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Biosciences, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Biosciences, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA. Electronic address: tpaull@utexas.edu.
authors.forename: Tanya T
authors.initials: TT
authors.lastname: Paull
mesh_terms: D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
chemical_list: D004277:DNA, Single-Stranded
keywords: DNA repair; Double-strand break repair; Resection; Single-stranded DNA