Quantitative analysis of genomic element interactions by molecular colony technique.

ABSTRACT: Distant genomic elements were found to interact within the folded eukaryotic genome. However, the used experimental approach (chromosome conformation capture, 3C) enables neither determination of the percentage of cells in which the interactions occur nor demonstration of simultaneous interaction of >2 genomic elements. Each of the above can be done using in-gel replication of interacting DNA segments, the technique reported here. Chromatin fragments released from formaldehyde-cross-linked cells by sodium dodecyl sulfate extraction and sonication are distributed in a polyacrylamide gel layer followed by amplification of selected test regions directly in the gel by multiplex polymerase chain reaction. The fragments that have been cross-linked and separate fragments give rise to multi- and monocomponent molecular colonies, respectively, which can be distinguished and counted. Using in-gel replication of interacting DNA segments, we demonstrate that in the material from mouse erythroid cells, the majority of fragments containing the promoters of active ?-globin genes and their remote enhancers do not form complexes stable enough to survive sodium dodecyl sulfate extraction and sonication. This indicates that either these elements do not interact directly in the majority of cells at a given time moment, or the formed DNA-protein complex cannot be stabilized by formaldehyde cross-linking.

SUBMITTER: Gavrilov AA 

PROVIDER: S-EPMC3950710 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature