Determination of DNA replication kinetics in synchronized human cells using 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 analysis. 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 content, at different times after synchronous release from the G1/S phase boundary. The selective removal of newly-replicated BrdU-substituted DNA was achieved by UV light irradiation followed by S1 nuclease treatment. The timing of replication of selected DNA sequences (housekeeping, tissue-specific, and non-coding loci) was determined by polymerase chain reaction (PCR) amplification using appropriate primers. DNA sequences localized in inactive replication units allowed amplification whereas 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.
Sinnett
D
D
Genetics Division, Children's Hospital, Boston, MA 02115.
Flint
A
A
Lalande
M
M
D001973:Bromodeoxyuridine; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000528:radiation effects; D004261:DNA Replication; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D016196:S Phase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014466:Ultraviolet Rays
D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases; D001973:Bromodeoxyuridine