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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 off-chip 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.

SUBMITTER: Benitez JJ

PROVIDER: S-EPMC3954578 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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

Benítez Jaime J JJ Topolancik Juraj J Tian Harvey C HC Wallin Christopher B CB Latulippe David R DR Szeto Kylan K Murphy Patrick J PJ Cipriany Benjamin R BR Levy Stephen L SL Soloway Paul D PD Craighead Harold G HG

Lab on a chip 20121101 22

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 w ...[more]

PMID: 23018789

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Project description:Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the extraction of cfDNA from a variety of clinical samples can be an effective and minimally invasive source of markers for disease detection and subsequent management. In the oncological diseases, circulating tumor DNA (ctDNA), a cfDNA sub-class, can carry clinically actionable mutations and coupled with next generation sequencing or other mutation detection methods provide a venue for effective in vitro diagnostics. However, cfDNA mutational analyses require high quality inputs. This necessitates extraction platforms that provide high recovery over the entire ctDNA size range (50 ? 150 bp) with minimal interferences (i.e., co-extraction of genomic DNA), and high reproducibility with a simple workflow. Herein, we present a novel microfluidic solid-phase extraction device (?SPE) consisting of a plastic chip that is activated with UV/O3 to generate surface-confined carboxylic acid functionalities for the ?SPE of cfDNA. The ?SPE uses an immobilization buffer (IB) consisting of polyethylene glycol and salts that induce cfDNA condensation onto the activated plastic microfluidic surface. The ?SPE consists of an array of micropillars to increase extraction bed load (scalable to loads >700 ng of cfDNA) and can be produced at low-cost using replication-based techniques. The entire ?SPE can be fabricated in a single molding step negating the need for adding additional extraction supports to the device simplifying production and keeping device and assay cost low. The ?SPE allowed for recoveries >90% of model cfDNA fragments across a range of sizes (100-700 bp) and even the ability to extract efficiently short cfDNA fragments (50 bp, >70%). In addition, the composition of the IB allowed for reducing the interference of co-extracted genomic DNA. We demonstrated the clinical utility of the ?SPE by quantifying the levels of cfDNA in healthy donors and patients with non-small-cell lung and colorectal cancers. ?SPE extracted cfDNA from plasma samples was also subjected to a ligase detection reaction (LDR) for determining the presence of mutations in the KRAS gene for colorectal and non-small cell lung cancer patients.

Dataset Information

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

Publications

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

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