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

Karata K, Vidal AE, Woodgate R
DNA repair (2009), Volume 8, Page 852
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Abstract:

We report a concise and efficient method to make a circular ...
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.

Polymerases:

T7

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