Abstract
A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and “time of replication” for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of ≤ ±10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5′-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
