We demonstrate experimentally that structural perturbations imposed on highly-

No abstract

We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for offchip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level.

Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing. I n chromatin, chemical modifications to histone proteins and DNA alter the status of the epigenome and influence gene regulation and normal development. Their aberrant placement has been linked to the onset of cancer (1, 2) and other diseases. Bisulfite conversion and immunoprecipitation (IP) have been used extensively to examine these modifications on locus-specific and genome-wide scales. These approaches have limitations in terms of material handling or multiplexed detection. Conventional chromatin immunoprecipitation (ChIP) requires an abundance of input material, often 10 3 -10 6 cells for genome-wide studies, to compensate for >99% material loss during processing (3, 4). This problem compounds for sequential re-ChIP reactions, limiting the study of multivalent modifications (4), which could provide a clear view of epigenetic coordination. Whereas DNA methylation analysis using bisulfite conversion can operate on picogram quantities of DNA (5-7), the conversion causes degradation of >90% of the input DNA. Methods that combine ChIP and bisulfite sequencing in a sequential process (8) have demonstrated progress in multiplexed epigenetic analysis. There continues to be active research in reducing the input material requirements and in automation of the processes (9-11) for epigenetic analysis. Furthermore, there is interest in additional capability for simultaneous detection of multiple epigenetic modifications in the same material.Miniaturized fluidic devices offer a compelling toolset for multiplexed detection and efficient sample handling in analytical and preparatory systems. Microfluidics have performed complicated workflows that include nanoliter sample handling (12) and incorporate electrodes (13-16) or valves (12, 17) for sophisticated processing. Nanofluidics have achieved attoliter-scale fluid volume confinement to isolate and quantify the attributes of individual molecules that can ...