Microfluidic-assisted analysis of replicating DNA molecules

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Abstract
Single molecule-based protocols have been gaining popularity as a way to visualize DNA replication at the global genomic- and locus-specific levels. These protocols take advantage of the ability of many organisms to incorporate nucleoside analogs during DNA replication, together with a method to display stretched DNA on glass for immunostaining and microscopy. We describe here a microfluidic platform that can be used to stretch and to capture labeled DNA molecules for replication analyses. This platform consists of parallel arrays of three-sided, 3- or 4-μm high, variable-width capillary channels fabricated from polydimethylsiloxane by conventional soft lithography, and of silane-modified glass coverslips to reversibly seal the open side of the channels. Capillary tension in these microchannels facilitates DNA loading, stretching and glass coverslip deposition from microliter-scale DNA samples. The simplicity and extensibility of this platform should facilitate DNA replication analyses using small samples from a variety of biological and clinical sources.