Forensic Typing of Single Cells Using Droplet Microfluidics

Abstract

Short tandem repeat (STR) typing based on polymerase chain reaction (PCR) is a powerful tool for forensic identification. However, it is challenging to analyze low-quantity and low-quality evidence samples comprising mixture of cells and/or DNA from multiple contributors. Herein we present a droplet microfluidics-based technology for single-cell STR typing with high sensitivity, throughput, and fidelity. In this approach, single cells are initially compartmentalized within nanoliter agarose droplets using a microfluidic droplet generator. The transformation of the agarose into microgels enables the diffusion of cell lytic reagents into the droplets for single-cell genomic DNA extraction, while PCR inhibitors are washed away. Following the introduction of PCR components, massively parallel multiplex droplet PCR is performed in oil emulsions to transfer the STR information from an individual cell onto the co-encapsulated primer-functionalized bead. The amplicon-carrying beads are then statistically diluted and reamplified in a secondary PCR. The resulting transfer of STR products to free solution facilitates the detection by conventional capillary electrophoresis (CE) fragment size analysis. This technology is effective in preserving single-genome integrity when analyzing diverse cellular materials yet insensitive to background DNA contamination. Target cells are successfully identified from heterogeneous cell populations with a high (up to 10:1) background, mixtures of cells and cell-free DNA, and samples contaminated with high concentrations of PCR inhibitors.