Use of the combing process for the identification of dna...

Details Use of the combing process for the identification of dna... Use of the combing process for the identification of dna...

C - Chemistry, Metallurgy

12

Q

C12Q 1/68 (2006.01)

Patent

CA 2375534

Eukaryotic genomes are duplicated by the activation of multiple bidirectional origins of replication. The replication programs of these cells depend on the temporal and spatial organisation of replication origins throughout the genome. To investigate the replication program in a higher eukaryote, we employed a technique called molecular combing. This technique allows for a quantitative analysis of DNA replication on a genome wide basis. As a model system, Xenopus laevis sperm chromatin were differentially labelled at successive time points after the beginning of DNA synthesis. Genomic DNA was then extracted and combed on a glass surface. Direct measurements made on the labelled DNA provided a comprehensive analysis of the spatial and temporal organisation of the X. leavis early embryo replication program and revealed that the number of replication origins activated per kilobase increases throughout the period of DNA synthesis.

La duplication des génomes eucaryotes se déroule par activation de plusieurs origines de réplication bidirectionnelles. Les programmes de réplication de ces cellules dépendent de l'organisation spatiale et temporelle de ces origines de réplication dans le génome. Afin d'étudier le programme de réplication chez un eucaryote supérieur, on a utilisé une technique appelée peignage moléculaire, qui permet l'analyse quantitative de la réplication d'ADN à l'échelle du génome. A cet effet, la chromatine du sperme de <i>Xenopus laevis</i>, choisi comme système modèle, a été marquée de manière différentielle en plusieurs points temporels successifs, après le début de la synthèse d'ADN. L'ADN génomique a ensuite été extrait et peigné sur une surface de verre. Les mesures directes effectuées sur l'ADN marqué ont fourni une analyse globale de l'organisation spatiale et temporelle du programme de réplication de l'embryon précoce du <i>X. laevis</i>, et révélé que le nombre d'origines de réplication activées par kilobase augmente tout au long de la synthèse d'ADN.

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