Construction of a circular single-stranded DNA template containing a defined lesion.
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.
Karata
Kiyonobu
K
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
Vidal
Antonio E
AE
Woodgate
Roger
R
D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D004249:DNA Damage; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D051981:Uracil-DNA Glycosidase / Q000378:metabolism
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; C020133:exodeoxyribonuclease III; D051981:Uracil-DNA Glycosidase