Direct measurement of single-stranded DNA intermediates in mammalian cells by quantitative polymerase chain reaction

作者：Zhou Yi; Paull Tanya T^*

来源：Analytical Biochemistry, 2015, 479: 48-50.

DOI：10.1016/j.ab.2015.03.025

• DNA repair
• Resection
• Single-stranded DNA
• Double-strand break repair

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.

• 出版日期2015-6-15