Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells

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.

Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells

“…There are several additional embodiments possible for our system that can expand its capabilities, including performing parallel analyses in multiple channels to increase throughput; adding a third fluorescent color, which would allow for absolute quantitation of epigenetic marks; and implementing a recently developed method to sort single DNA molecules based on epigenetic state (35) which would allow for downstream sequencing. Because individual molecules are queried, only small amounts of cellular material are required, raising the possibility of epigenomic analyses of cells that are rare, impossible to culture, or even of single cells (36).…”

Micro- and nanoscale devices for the investigation of epigenetics and chromatin dynamics

“…Methods have been developed over the last decade to isolate single cells from an ensemble using miniaturized systems that take advantage of novel physical phenomena and intrinsic cellular properties 112 . These microfluidic isolation techniques can be combined with new approaches to perform extraction of intact chromosomes 113,114 from a single cell 115 , partitioning of each chromosome into a small volume chamber 116 and interrogation using one or several of the micro/nano-based techniques described above, followed by sorting specific molecules of interest 78,79 and sequencing the extracted samples (Figure 5). Microfluidic integration also permits streamlined assays with precisely controlled inputs such as concentration gradients, temperature changes and serial (or parallel) introduction of stimuli.…”

“…In the first stage of the device (blue channels), specific cells of interest are microfluidically isolated 112 from a population and a single cell is trapped in a small-volume chamber. Next, in-tact chromosomes are extracted from the cell 113,114,115 and partitioned (green channels) into chambers where each is profiled for multiple epigenetic modifications using one or several of the micro/nano-based techniques (orange channels). Once profiled, specific molecules of interest can then be sorted 78,79 and recovered (white channels) for amplification or sequencing.…”

Measuring gene expression in single bacterial cells: recent advances in methods and micro-devices

“…This technology is especially advantageous to the manipulation and analysis at the single-cell level for the following reasons: (i) Individual cells can be precisely trapped, moved, and distributed individually in microscale channels; (ii) Isolated individual cells can be easily monitored in microchambers and dilution of the cellular contents is minimized thus increasing the sensitivity of a downstream analysis; and (iii) Highly parallel, fully automated multi-step operations can be implemented for high-throughput analyses resulting in significant time and cost savings. The application of microfluidic devices to study single eukaryotic cells has been a thriving field (Banaeiyan et al, 2013;Benítez et al, 2012;Beta & Bodenschatz, 2011;Clausell-Tormos et al, 2008;Meldrum & Holl, 2002;Molter et al, 2009Molter et al, , 2008Roman et al, 2007;White et al, 2011;Zhu et al, 2012). While applying these technologies to microbiology research is at its early age, rapid developments have been witnessed in recent years.…”