Mammalian DNA replication initiates at multiple sites along chromosomes at 
different times during S phase, following a temporal replication program. The 
specification of replication timing is thought to be a dynamic process regulated 
by tissue-specific and developmental cues that are responsive to epigenetic 
modifications. However, the mechanisms regulating where and when DNA replication 
initiates along chromosomes remains poorly understood. Homologous chromosomes 
usually replicate synchronously, however there are notable exceptions to this 
rule. For example, in female mammalian cells one of the two X chromosomes 
becomes late replicating through a process known as X inactivation(1). Along 
with this delay in replication timing, estimated to be 2-3 hr, the majority of 
genes become transcriptionally silenced on one X chromosome. In addition, a 
discrete cis-acting locus, known as the X inactivation center, regulates this X 
inactivation process, including the induction of delayed replication timing on 
the entire inactive X chromosome. In addition, certain chromosome rearrangements 
found in cancer cells and in cells exposed to ionizing radiation display a 
significant delay in replication timing of >3 hours that affects the entire 
chromosome(2,3). Recent work from our lab indicates that disruption of discrete 
cis-acting autosomal loci result in an extremely late replicating phenotype that 
affects the entire chromosome(4). Additional 'chromosome engineering' studies 
indicate that certain chromosome rearrangements affecting many different 
chromosomes result in this abnormal replication-timing phenotype, suggesting 
that all mammalian chromosomes contain discrete cis-acting loci that control 
proper replication timing of individual chromosomes(5). Here, we present a 
method for the quantitative analysis of chromosome replication timing combined 
with fluorescent in situ hybridization. This method allows for a direct 
comparison of replication timing between homologous chromosomes within the same 
cell, and was adapted from(6). In addition, this method allows for the 
unambiguous identification of chromosomal rearrangements that correlate with 
changes in replication timing that affect the entire chromosome. This method has 
advantages over recently developed high throughput micro-array or sequencing 
protocols that cannot distinguish between homologous alleles present on 
rearranged and un-rearranged chromosomes. In addition, because the method 
described here evaluates single cells, it can detect changes in chromosome 
replication timing on chromosomal rearrangements that are present in only a 
fraction of the cells in a population.