Microfluidic-assisted analysis of replicating DNA molecules.
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-microm 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.
Sidorova
Julia M
JM
Department of Pathology, University of Washington, Seattle, Washington, USA. julias@u.washington.edu
Li
Nianzhen
N
Schwartz
David C
DC
Folch
Albert
A
Monnat
Raymond J
RJ
D002874:Chromosome Mapping / Q000379:methods; D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D004261:DNA Replication; D005796:Genes; D023281:Genomics / Q000379:methods; D005898:Glass; D007202:Indicators and Reagents; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D008904:Miniaturization / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012825:Silicon
D007202:Indicators and Reagents; D004247:DNA; D012825:Silicon