Replication Combing Assay (RCA)
For spatiotemporal analysis of DNA replication
Direct visualization of active origins and studies of replication kinetics on long DNA molecules
Address various questions about the replication program in a single experiment
Study initiation rates (activated origins/total length of DNA molecules)
Measure fork velocity and fork progression
When combined with fluorescence in-situ hybridization, analyze activated origin patterns and kinetics at a region of interest:
Map active origins
Measure origin efficiency
Dynamic Studies of DNA Replication & Repair
Molecular Combing is a powerful and accurate approach to monitor both spatial and temporal characteristics of DNA replication in a single experiment. On combed DNA, replication can be directly visualized using immunofluorescence techniques. This involves dual-pulse labelling of the replicating DNA with modified nucleotides. Depending on the labelling scheme used, earlier and later replicating sequences in each molecule can be distinguished as alternating green and red fluorescent segments.
We have developed an experimental protocol, the Replication Combing Assay for the analysis of replication profiles on DNA molecules of comparable length. This unique assay allows for studying overall origin distributions and fork elongation rates during normal or abnormal cell division. Multiple initiation sites on the same DNA molecule can be both spatially and temporally monitored, directly on combed DNA.
The replication program occurring at specific genetic locus can be determined by simultaneously carrying out fluorescent detection of hybridized probes for the region of interest and of incorporated modified nucleotides. Our standardized protocol used with the Molecular Combing Platform allows for reproducible studies and delivers accurate and fast results.
How it works
Replicating cells are incubated with halogenated nucleotides. At specific times during the incubation, samples of cells are removed and their DNA is isolated and subjected to Molecular Combing before undergoing fluorescent probe hybridization and analysis.
Schematic representation of signals deriving from equal pulse-labeling with iodo-deoxyuridine (IdU) and chloro-deoxyuridine (CldU)
Replication forks progress bidirectionally at the same rate from the origin and incorporate the analogues forming a symmetrical replication bubble. On detection of IdU and CldU, three types of signals may be obtained:
(1) initiations that occurred before the beginning of the pulse:
a green and red signal with a gap between the green segments
(2) origins that fired during the first pulse:
a dual-color signal with a continuous green segment
(3) origins that fired during the second pulse:
a single isolated red signal
A continuous red signal flanked by two green ones is formed by the merging of two forks from adjacent origins.
Applications for Cancer Drug Discovery & Testing efficacy of Lead Compounds
A comprehensive analysis of replication profiles under chosen genetic and physiological conditions can facilitate the identification of promising drugs for use in cancer therapy. By using the Replication Combing Assay to evaluate the efficacy of anti-cancer molecules and to detect genomic instability, pharmaceutical companies can also have access to a novel, high-performance approach for testing lead compounds.