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.
Publication types
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Research Support, N.I.H., Intramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Bacteriophages / genetics
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Base Sequence
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DNA Damage*
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DNA Replication
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DNA, Circular / genetics*
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DNA, Circular / metabolism
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DNA, Single-Stranded / genetics*
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DNA, Single-Stranded / metabolism
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Escherichia coli Proteins / metabolism
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Exodeoxyribonucleases / metabolism
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Genetic Techniques
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Genetic Vectors / genetics
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Molecular Sequence Data
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Uracil-DNA Glycosidase / metabolism
Substances
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DNA, Circular
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DNA, Single-Stranded
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Escherichia coli Proteins
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Exodeoxyribonucleases
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exodeoxyribonuclease I
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exodeoxyribonuclease III
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Uracil-DNA Glycosidase