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Single cell genetic analysis using nanoliter emulsion droplet technology offers the ultimate in digital sensitivity and an unprecedented view of the characteristics of cell populations that are masked by the ensemble average. In many cancers, small subpopulations of distinct cell types cause or drive the disease but are difficult to study using conventional biomolecular methods. In forensics, the ability to type individual cells and to analyze cellular mixtures cell-by-cell offers unprecedented identification capabilities. To enable this new analysis technology, we have developed microfluidic droplet generators that produce highly uniform 1-5 nL droplets at very high production rates that are subsequently used to perform PCR and RT-PCR analyses. A significant challenge is the release of genomic DNA from single cells in these nanoliter droplets. This challenge has been addressed by encapsulating cells together with primer functionalized beads in nanoliter agarose droplets which can then be used as porous carriers for sequentially performing cell lysis and cleanup followed by microemulsion PCR. The subsequent analysis of the individual beads by flow cytometry, capillary electrophoresis or sequencing techniques then reveals the target presence, size or sequence, as desired. Specifically, in one application we have developed a method for high-throughput single cell DNA purification and detection of the translocation, an approach for investigating the frequency of rare mutation events within individuals prior to the onset of clinical symptoms. In a second application, methods have been developed for performing a 9-plex short tandem repeat amplification in the nanoliter emulsion droplets that provides high fidelity forensic typing of single cells in cellular mixtures. These single cell techniques enable the quantitative study of cellular behavior with unprecedented sensitivity that should lead to a better mechanistic understanding of cellular processes such as cancer onset and progression. The application of single-cell analysis to forensics should similarly revolutionize human identification with digital sensitivity and unprecedented mixture analysis capabilities.
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