Quantification of Replication Origins

Genomic DNA from 1 OD₆₃₀ of cells was extracted and purified as previously described.⁸² (link) 3 ng of DNA was used per qPCR reaction (qPCR Master Mix, NEB). PCR was performed using an Applied Biosystems 7500 instrument (software version 2.3). PCR primers are listed in Table 4. Briefly, qPCR signal for a given origin was first normalized to the signal obtained from the NegV locus (ChrV: 532538-532516).⁶⁹ (link) This region is located ≈12 kb from ARS521, an origin which has not been detected to be active in several studies according to OriDB (http://cerevisiae.oridb.org/) and ≈18 kb from ARS522, a subtelomeric origin of replication activated in late S. As such, the NegV locus is expected to be replicated in late S, and therefore to generally remain unreplicated in a majority of cdc7-4 and cdc7-4 hst3Δ hst4Δ cells 30 min post-release from G1 arrest toward S phase. The NegV-normalized S phase signal was divided by the NegV-normalized signal obtained from alpha factor arrested (G1) cells. Complete replication of an origin is therefore expected to result in a ratio of S phase over G1 signal of 2.