Analyzing origin activation patterns by copy number change experiments

Miruthubashini Raveendranathan, Anja Katrin Bielinsky

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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 ~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.

Original languageEnglish (US)
Title of host publicationDNA Replication
Subtitle of host publicationMethods and Protocols
Pages279-294
Number of pages16
DOIs
StatePublished - 2009

Publication series

NameMethods in Molecular Biology
Volume521
ISSN (Print)1064-3745

Keywords

  • Copy number
  • DNA microarrays
  • Origin activation
  • Replication origins
  • S phase
  • Yeast

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