DETERMINATION OF DNA-REPLICATION KINETICS IN SYNCHRONIZED HUMAN-CELLSUSING A PCR-BASED ASSAY

Studies on the temporal order of DNA replication are difficult due to the lack of sensitivity of methods available for replication kinetic a nalysis. To overcome problems associated with the current techniques, we propose a PCR-based assay to determine the replication time of any single-copy DNA sequence in complex genomes. Human cells labeled with 5-bromodeoxyuridine (BrdU) were flow sorted, according to their DNA co ntent, at different times after synchronous release from the G1/S phas e boundary. The selective removal of newly-replicated BrdU-substituted DNA was achieved by UV light irradiation followed by S1 nuclease trea tment. The timing of replication of selected DNA sequences (housekeepi ng, tissue-specific, and non-coding loci) was determined by polymerase chain reaction (PCR) amplification using appropriate primers. DNA seq uences localized in inactive replication units allowed amplification w hereas those that have replicated will not be amplified by PCR. Using this sensitive and quantitative assay the replication kinetic analysis of a number of different DNA sequences can be performed from a single sorting experiment.