Rapid high-resolution measurement of DNA replication timing by droplet digital PCR

Dzmitry G. Batrakou, Emma D. Heron, Conrad A. Nieduszynski

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4 Citations (Scopus)

Abstract

Genomes are replicated in a reproducible temporal pattern. Current methods for assaying allele replication timing are time consuming and/or expensive. These include high-throughput sequencing which can be used to measure DNA copy number as a proxy for allele replication timing. Here, we use droplet digital PCR to study DNA replication timing at multiple loci in budding yeast and human cells. We establish that the method has temporal and spatial resolutions comparable to the high-throughput sequencing approaches, while being faster than alternative locusspecific methods. Furthermore, the approach is capable of allele discrimination. We apply this method to determine relative replication timing across timing transition zones in cultured human cells. Finally,multiple samples can be analysed in parallel, allowing us to rapidly screen kinetochore mutants for perturbation to centromere replication timing. Therefore, this approach is well suited to the study of locus-specific replication and the screening of cis- andtrans-acting mutants to identify mechanisms that regulate local genome replication timing.

Original languageEnglish
Article numbere112
JournalNucleic Acids Research
Volume46
Issue number19
Early online date9 Jul 2018
DOIs
Publication statusPublished - 2 Nov 2018

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