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MDC allows the access to the molecular combing technique for single-molecule analysis of DNA replication along eukaryotic chromosomes. This facility is unique in the academic world and attracts national and international clients. Molecular combing allows stretching and uniformly aligning long DNA fibres on silanized slides. These fibres can then be analysed by fluorescence microscopy (FISH, immunofluorescence).
Application Portfolio:
- Study of chromosomal micro-rearrangements,
- Fine study of the chromosome replication dynamics. Replicating cells are pulsed with thymidine analogues (IdU, CldU). Then, specific antibodies coupled to different fluorophores are used to detect the incorporation of these analogues along combed single DNA molecules that can reach 2 Mb in length. The mean distances between DNA replication origins, their global density, the speed as well as the progression defects of replication forks are determined quantitatively, with high resolution, genome-wide, but in single molecules.
A crucial feature of molecular combing, compared with the other available analysis techniques, is that it does not average the values obtained in a population. This allows, for instance, the detection of subtle replication defects that cannot be observed with other means.
Main expertise/Services:
- Production, quality control, and distribution of silanized glass slides
- Complete biological analyses (from DNA preparation to the quantification of the replication dynamic parameters and statistical tests), in collaboration
- Analysis of molecular combing images with our dedicated software IDeFIx, created by the facility staff
- Training in the molecular combing technique (from slide silanization, cell labelling, DNA preparation and combing, to the informatics analysis of the obtained data).
Access and service policy:
Silanized slides are provided only to CNRS laboratories. The other services are open to all users (public- and private-sector). We carry out complete DNA replication analyses on a collaborative basis. The other services are fee-based.
Publications
Applications note:
