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. Author manuscript; available in PMC: 2021 Feb 1.
Published in final edited form as: DNA Repair (Amst). 2019 Dec 5;86:102766. doi: 10.1016/j.dnarep.2019.102766

Figure 1. Method for detecting sedDNAs in human skin epidermis.

Figure 1.

(a) Schematic of the nucleotide excision repair mechanism, in which UVB-damaged bases (indicated by thymine dimer, T<>T) are removed by via dual incisions to generate a sedDNA. (b) Sample workflow for skin processing and detection of the sedDNA products of NER. Small, 6 mm punch biopsies of discarded human skin were obtained from UVB-irradiated skin and snap frozen. Epidermal tissue was removed from the biopsy by heat shock and lysed. Soluble DNAs obtained from the epidermal lysates were then immunoprecipitated with (6-4)PP antibody, 3’ end-labeled with a biotinylated nucleotide, electrophoresed on a polyacrylamide gel, transferred to a nylon membrane, and then detected with HRP-coupled streptavidin and chemiluminescence. (c) The image shows a typical experimental result from skin harvested 1 hr after exposure to 800 J/m2 of UVB radiation. sedDNA labeling reactions were supplemented with a 50-mer ssDNA oligonucleotide standard. Location of DNA size markers (nt) is indicated.