PH stepwise alkaline elution of DNA replication intermediates during S phase

We have used a newly developed pH stepwise alkaline elution method to analyze the distribution of DNA replication intermediates during S phase. Using synchronized CHO (Chinese Hamster Ovary) cells, replicon-size replication intermediates (≤ 15S) reach a maximum in the first half of S phase. These intermediates are functional in that they are used to generate larger DNA fragments. The larger DNA fragments corresponding in size to replicating sections increase linearly with the progression of cells throughout S phase, corresponding to DNA replication kinetics previously obtained by CsCl density gradient analysis. These data will enable us to enrich for particular DNA replication fragments and identify the protein components associated with these intermediates.