Analyzing origin activation patterns by copy number change experiments.
Advances in microarray technology have enabled the analysis of replication dynamics on a genome-wide scale, providing deeper insight to the factors that regulate DNA replication. Studies using high-density microarrays have led to the genome-wide identification of replication origins in the budding yeast, Saccharomyces cerevisiae, and enabled the analysis of the global temporal pattern of origin activation under various conditions. We have developed a replication origin array that contains the approximately 430 potential origins in the yeast genome. By detecting the copy number change that occurs as cells progress from G1 to S phase on these arrays, we have produced origin activation patterns in wild-type cells similar to those obtained from previous studies that used whole-genome arrays. We have also applied this method to study S phase checkpoint mutants, providing insight into the genome-wide regulation of replication origin activation by S phase checkpoint kinases in the presence of replication stress. The main procedures of this technique involve arresting yeast cells in G1 and S phase, isolating and labeling genomic DNA with fluorescent dyes, and cohybridizing the DNA samples to replication origin arrays to yield copy number change data.
Raveendranathan
Miruthubashini
M
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
Bielinsky
Anja-Katrin
AK
D015825:Chromosomes, Fungal / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016193:G1 Phase / Q000235:genetics; D018628:Gene Dosage; D016681:Genome, Fungal; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D018741:Replication Origin; D016196:S Phase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal