NUCLEOTIDE LEVEL DETECTION OF CYCLOBUTANE PYRIMIDINE DIMERS USING OLIGONUCLEOTIDES AND MAGNETIC BEADS TO FACILITATE LABELING 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 inci sed at the CPDs with Micrococcus luteus UV endonuclease. The subsequen t specific fragments were separated using a biotin labelled oligonucle otide containing a sequence complementary to the fragments of interest and streptavidin magnetic beads. These fragments were then radiolabel led 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 trea tments. These chemically cleaved fragments can be stored almost indefi nitely, Whenever the sequence reference ladders are required, the chem ically cleaved fragments can be labelled alongside the CPD-specificall y incised DNA fragments using the same procedure, The adaptation of th e method to detect other types of DNA damage is also discussed.