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Single-Cell Multiplex Gene Detection and Sequencing with Microfluidically Generated Agarose Emulsions

Center for Exposure Biology, University of California, Berkeley, 307 Lewis Hall, Berkeley, CA 94720, USA.
Angewandte Chemie International Edition (Impact Factor: 11.26). 01/2011; 50(2):390-5. DOI: 10.1002/anie.201006089
Source: PubMed

ABSTRACT Genetic assays, such as polymerase chain reaction (PCR), typically report on multiple cells or mixtures of genomic DNA. As a result, they cannot properly characterize the genetic heterogeneity of a cell population or detect the cooccurrence of different mutations within a single cell; these factors are key to understanding the development, progres-sion, and treatment of cancers. [1, 2] In particular, since initial mutagenesis occurs inherently at the single-cell level, the detection and characterization of carcinogenesis will be dramatically facilitated by analytical techniques with single-cell resolution. Cytometric sorting, limiting dilution, and micromanipu-lation have been previously used to perform single-cell PCR assays in 96-well PCR plates, but these approaches are not ideal for large-scale screening applications. [3] Microfluidic technology offers fundamentally new capabilities for the manipulation of fluids, molecules, and cells that are very pertinent for the development of high-throughput single-cell analysis methods. [4–7] Microfluidic droplet technology is particularly advantageous for single-cell/molecule analysis because it facilitates the rapid statistical compartmentaliza-tion of targets for massively parallel pico-to nanoliter-scale assays. [8–10] In particular, microfluidic emulsion PCR (ePCR) enables high-fidelity digital single-molecule counting owing to its unique ability to ensure equal population sampling and amplification efficiencies across all reaction compart-ments. [4, 11, 12] To date, most reported single-cell genomic analyses have been carried out on bacterial samples. [13–15] For mammalian cells, droplet-based genetic analyses have predominantly implemented reverse transcriptase PCR for phenotypic profiling. [11, 16] A difficulty in single-cell PCR is the persistent technical challenge of integrating a robust and scalable DNA-extraction method. [13, 14] The relative lack of suitable technol-ogies for single-cell genomic analysis combined with the significant genetic heterogeneity associated with cancer underscores the importance of developing new microdroplet methodologies that integrate robust single-cell genome preparation with multiplex PCR. To address these challenges, we have developed an agarose-droplet-based platform that leverages emulsion-gen-erator-array technology for high-throughput single-cell genetic analysis. [4, 12] Single cells were microfluidically encap-sulated together with primer-functionalized beads in agarose-gel droplets for subsequent SDS lysis and proteinase K digestion to release genomic DNA. With the coencapsulated primer beads and purified genomes, we demonstrate multi-locus single-cell sequencing of the control gene b-actin and the chromosomal translocation t(14;18), a mutation associ-ated with 85–90 % of cases of follicular lymphoma. [1, 17] The coupling of our robust and high-throughput single-cell DNA-purification method with the sequencing of multiple gene targets within single cells will enable detailed studies of mutation cooccurrence and synergy during carcinogenesis. The underlying principle of our highly parallel cell-digestion and DNA-purification method is the microfluidic encapsulation of cells in agarose droplets (Figure 1) to maintain single-genome fidelity during cell lysis and DNA purification as well as efficient multiplex emulsion PCR target amplification for subsequent analysis. Single lympho-blast cells were encapsulated along with primer-functional-ized beads in 1.5 % low-melting-point agarose by using a four-channel microfluidic-emulsion-generator array (MEGA; Fig-ure 2 a). [4] The MEGA platform is very versatile: micropump Figure 1. Workflow diagram showing the use of agarose-emulsion droplets for the genetic analysis and multilocus sequencing of single mammalian cells. a) Single cells are microfluidically encapsulated together with primer-functionalized beads in agarose-gel droplets. b) The genomes of single cells are released in the gel droplets upon SDS lysis and digestion with proteinase K according to a standard protocol. c) The agarose droplets are equilibrated in PCR buffer containing fluorescent forward primers, emulsified with oil by mechan-ical agitation, and thermally cycled. d) Following multiplex PCR amplifi-cation, primer beads are released by breaking the emulsion and melting the agarose. The fluorescent beads are then rapidly quantified by flow cytometry or further subjected to PCR amplification for the sequencing of target genes. SDS = sodium dodecyl sulfate.

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    • "Benefited from abundant manipulation strategies of acoustic [14], optical [15], thermal [16], magnetic [17], valve-based [18], hydrodynamic [19] and electric [20] methods, droplets can be split, mixed, transported, trapped, sorted etc. And after dispersing cells into nano-liter or pico-liter droplets, combining aforementioned droplet microfluidic techniques can thus fulfill various demands for cell-based research and applications at single cell level in these nano laboratories, including immunoassays [21] [22], 3D cell culture and division [23] [24], cell sorting [25], cytokine secretion [26], gene detection and sequencing [27] [28], drug testing [29], DNA amplification [30] and so on. Although multiple physical manipulations are employed to expand the applications of droplet microfluidics in single cell research, there is still a long way for them to be comprehensively used. "
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    • "(a) Agarose droplet-based single-cell ddPCR system. (Reproduced from [27], with permission from Wiley). (b) Water-in-oil droplet-based workflow platform for single-cell ddPCR, where drop compartmentalization is maintained at all times. "
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    • "Although arrays for droplet trapping have been developed for time scale study of single cells [38] [40] [41], these do not allow for the retrieval of cells of interest to a macro scale accessible format. Fluorescence activated cell sorting (FACS) is a method widely used in the biological community for high throughput cell sorting and biomarker detection, which can be used as a chip-to-world output format [42]. Compared to FADS, state-of-the-art FACS provides at least an order of magnitude higher throughput, but it lacks the compartmentalization of biological components offered by droplet microfluidics [43]. "
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