Skip to main content
. 2006 May;17(5):2401–2414. doi: 10.1091/mbc.E05-11-1043

Figure 1.

Figure 1.

Use of comparative genomic hybridization (CGH) on spotted microarrays to assay DNA replication. (A) Schematic representation of the CGH replication assay. Genomic DNA is purified from nonreplicating and replicating cells, differentially labeled with Cy3 and Cy5, and competitively hybridized to a microarray containing 12,034 ORF and intergenic PCR products. Cy5/Cy3 ratios are normalized so that the average ratio of all elements equals the DNA content of the cells (as determined by flow cytometry). Normalized ratios are plotted against chromosomal position and mathematically smoothed to generate a replication profile. In most cases, two hybridizations are performed from each of two independent experiments. The resulting four replication profiles are averaged into one composite profile, and the locations of origins are identified using a peak finding algorithm. Chromosomal regions lacking data of sufficient quality are represented as gaps in the profiles. (B) CGH replication assay described for A was performed on YJL5038, a wild-type yeast strain in the S288c background. G1 phase genomic DNA was hybridized against S phase genomic DNA obtained 120 min after cells were released from G1 phase into media containing hydroxyurea (HU). The composite replication profile (blue line) plus and minus the “experiment variability” (light gray band; see Materials and Methods) is shown for chromosome X. Positions of origins annotated in the Saccharomyces Genome Database (SGD; Balakrishnan (2006); red triangles) and the centromere (black circle) are marked along the X-axis. Replication profiles derived from Raghuraman et al. (2001) (violet line) and Yabuki et al. (2002) (orange line) are shown for comparison. (C) S phase progression assayed by flow cytometry for experiment described in B at the indicated times after release from G1 phase. DNA content of 1.4 C was used to normalize the S288c replication profile. (D) The S phase replication profile of the re-replication competent OMC strain and the congenic wild-type strain are similar. S phase replication profiles were generated for the OMC strain YJL3248 (MCM7-2NLS orc2-cdk6A orc6-cdk4A pGAL1-Δntcdc6 pMET3-HA3-CDC20) and a congenic wild-type A364a strain YJL5834 (pGAL1) essentially as described in B except S phase cells were harvested, respectively, at 135 min and 180 min after α factor release. The S phase replication profile for the OMC strain (green line) and the A364a strain (black line) for chromosome X is shown. SGD annotated origins (red triangles) and the centromere (black circle) are marked along the X-axis. (E) S phase progression assayed by flow cytometry for experiment described in D at the indicated times after release from G1 phase. DNA contents of 1.35 C and 1.4 C, respectively, were used to normalize the OMC and A364a replication profiles.