RESUMO
Single cell whole genome amplification is susceptible to amplification biases that impact the accuracy of single cell sequencing data. To address this, we have developed a microfluidic device for the isolation and purification of genomic DNA from individual cells. The device uses a micropillar array to physically capture single cells and its chromosomal DNA upon extraction. The extracted DNA is immobilized within the micropillar array in a way that allows isothermal amplification. In this system, whole genome amplification of the single cell is carried out under a continual fluid flow within the microfluidic channel. We have demonstrated the process for amplification of individual human cancer cell genomes from the HeLa cell line. By sampling select gene loci along the human genome and performing whole exome sequencing, we demonstrate improved genome coverage and reduced amplification bias compared to amplification of single cells deposited in wells by fluorescence activated cell sorting.
Assuntos
Genoma Humano , Dispositivos Lab-On-A-Chip , Reação em Cadeia da Polimerase/métodos , Citometria de Fluxo , Células HeLa , HumanosRESUMO
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.
Assuntos
Fracionamento Químico/instrumentação , Cromossomos Humanos/genética , DNA/análise , DNA/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única/instrumentação , Linhagem Celular Tumoral , DNA/química , Humanos , Conformação de Ácido Nucleico , Hibridização de Ácido NucleicoRESUMO
Individual chromatin molecules contain valuable genetic and epigenetic information. To date, there have not been reliable techniques available for the controlled stretching and manipulation of individual chromatin fragments for high-resolution imaging and analysis of these molecules. We report the controlled stretching of single chromatin fragments extracted from two different cancerous cell types (M091 and HeLa) characterized through fluorescence microscopy and atomic force microscopy (AFM). Our method combines soft lithography with molecular stretching to form ordered arrays of more than 250,000 individual chromatin fragments immobilized into a beads-on-a-string structure on a solid transparent support. Using fluorescence microscopy and AFM, we verified the presence of histone proteins after the stretching and transfer process.