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Construction of a circular single-stranded DNA template containing a defined lesion |
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| Authors: | Kiyonobu Karata Antonio E Vidal Roger Woodgate |
| Affiliation: | 1. Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy;2. Institute of Translational Pharmacology, National Research Council, CNR, Via Del Fosso del Cavaliere 100, 00133 Rome, Italy;1. Department of Biology and Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy;2. Institute of Translational Pharmacology, National Research Council, CNR, Via Del Fosso del Cavaliere 100, Rome 00133, Italy |
| Abstract: | We report a concise and efficient method to make a circular single-stranded DNA containing a defined DNA lesion. In this protocol, phagemid DNA containing Uracil is used as a template to synthesize a complementary DNA strand using T7 DNA polymerase and an oligonucleotide primer including a site-specific DNA lesion. The ligated lesion-containing strand can be recovered after the phage-derived template DNA is degraded by treatment with E. coli Uracil DNA glycosylase and Exonucleases I and III. The resulting product is a circular single-stranded DNA containing a defined DNA lesion suitable for in vitro translesion replication assays. |
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