Figure 2: Experimental strategy to determine the enrichment of short nascent DNA using DNA microarrays. (A) A hypothetical chromosome region undergoing DNA replication through points A, B, and C will produce nascent strand DNA of increasing size. The shortest DNA, which includes point B, will contain an initiation site for DNA replication. (B) Total DNA isolated from synchronously growing cells is isolated and the nascent strand DNA released by heat denaturation is size fractionated through a 5-30% sucrose gradient. Fractions containing DNA in the 0.7-1.5kb size range are pooled and treated with λ exonuclease. A similar size range DNA is produced by sonication of total DNA. Both DNA’s are differentially labeled and hybridized to a custom made high density tiling DNA array. By calculating the hybridization ratios at each one of the probes contained in the array a nascent DNA enrichment peak can be positioned within the genomic regions represented in the array.