Construction of a circular single-stranded DNA template containing a defined lesion

DNA Repair (Amst). 2009 Jul 4;8(7):852-6. doi: 10.1016/j.dnarep.2009.03.006. Epub 2009 Apr 21.


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.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophages / genetics
  • Base Sequence
  • DNA Damage*
  • DNA Replication
  • DNA, Circular / genetics*
  • DNA, Circular / metabolism
  • DNA, Single-Stranded / genetics*
  • DNA, Single-Stranded / metabolism
  • Escherichia coli Proteins / metabolism
  • Exodeoxyribonucleases / metabolism
  • Genetic Techniques
  • Genetic Vectors / genetics
  • Molecular Sequence Data
  • Uracil-DNA Glycosidase / metabolism


  • DNA, Circular
  • DNA, Single-Stranded
  • Escherichia coli Proteins
  • Exodeoxyribonucleases
  • exodeoxyribonuclease I
  • exodeoxyribonuclease III
  • Uracil-DNA Glycosidase