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

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

Author information

1
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.

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.

PMID:
19386556
PMCID:
PMC2700212
DOI:
10.1016/j.dnarep.2009.03.006
[Indexed for MEDLINE]
Free PMC Article

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