Nucleotide level detection of cyclobutane pyrimidine dimers using oligonucleotides and magnetic beads to facilitate labelling of DNA fragments incised at the dimers and chemical sequencing reference ladders.
We present a method for detecting cyclobutane pyrimidine dimers (CPDs) at the nucleotide level and an adaptation of Maxam-Gilbert sequencing for generating sequence reference ladders. UV irradiated genomic DNA from Escherichia coli was digested with restriction enzyme(s) and incised at the CPDs with Micrococcus luteus UV endonuclease. The subsequent specific fragments were separated using a biotin labelled oligonucleotide containing a sequence complementary to the fragments of interest and streptavidin magnetic beads. These fragments were then radiolabelled on the beads just prior to the running of the sequencing gel. For generating sequence reference ladders, the unlabelled DNA fragments of interest were base-specifically modified and subsequently cleaved at the A+G or C+T sites using the rapid Maxam-Gilbert sequencing treatments. These chemically cleaved fragments can be stored almost indefinitely. Whenever the sequence reference ladders are required, the chemically cleaved fragments can be labelled alongside the CPD-specifically incised DNA fragments using the same procedure. The adaptation of the method to detect other types of DNA damage is also discussed.
Li
S
S
Molecular Biology Group, School of Biological Sciences, University of Wales Swansea, UK.
Waters
R
R
D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects; D008280:Magnetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry*; D011740:Pyrimidine Dimers / Q000032:analysis*; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D009841:Oligonucleotides; D011740:Pyrimidine Dimers