Determination of the timing of replication of genetic markers by PCR. The three composite panels illustrate the procedure used for determining which of seven samples of DNA, each replicated at a different 1-h period of the S phase, was enriched for copies of a particular marker. DNA replicated at the indicated 1-h intervals of the S phase of synchronized fibroblasts was labeled with BrdUrd and isolated by CsCl centrifugation. In each panel, A: Inverted contrast image of PCR products stained with ethidium bromide after gel electrophoresis. The same primer set was used to amplify increasing amounts of genomic DNA and DNA replicated during each of the first 7 h of the S phase; a control with no template DNA was included in every PCR experiment. The R2 value for each standard curve that was obtained by plotting the signal intensity of the bands of PCR products in the gel above versus the amount of genomic DNA, is shown. B: Bar graph illustrating the abundance of the marker in each of the seven 1-h samples of the S phase. Results show the average of two synchronizations that were each tested at least twice. Vertical bars indicate the standard deviation for each time point. Relative abundance was calculated from the linear regression equation of the standard curve and expressed as a percentage of the S phase fraction with the highest value. Replication times were calculated as described in the Methods section. The three panels shown illustrate the results obtained with markers that replicate in the first hour of the S phase (primer chr22.1096B), during the second hour (chr6.1385B), and at 4.6 hrs into S phase (chr6.1417A).