Replication timing analysis

The replication timing assay is a current lab method used to determine the exact phases of replication process. Exponentially growing cells are labelled with 5- bromo 2-deoxyuridine to mark newly generated cells. Indeed, the bromo-deoxyuridine is incorporated into DNA in place of thymidine. Fluorescent dye DAPI stains the whole cellular DNA and enables to fractionate cells on the basis of total amount of DNA and thus, the cells’ stage of the synthesis phase of cell division.
Then, labelled DNA is isolated by immunoprecipitation and analysed by PCR on the loci of interest. A recent paper published this year in PNAS, proposes to substitute the PCR with the DNA assembling into separate Illumina sequencing libraries and sequence it. It seems that the most of the genome is going to be replicated at about the same time in different cell types, but the study of other cells populations will be clarified the timing. Furthermore, sequencing will permit analysis also of rare cell populations because of the low number of cells required for the assay. In conclusion, this new approach of replication timing assay allows extending many analyses at the whole genome levels, improving the throughput and accelerating scientific advancement.