Microchip Analyzer for Forensic Short Tandem Repeat Typing of Single Cells

Short tandem repeat (STR) typing at the single-cell level is a promising tool for human forensic identification when the biological evidence materials are comprised of mixtures of cells from multiple individuals at relatively low concentrations. Here we describe a novel single-cell STR typing method with high sensitivity, fidelity and throughput that combines microfluidic droplet generation with single-cell multiplex emulsion PCR. Individual cells are separately isolated within microdroplets that subsequently function as miniaturized reactors for PCR amplification, producing high quality STR profiles from single cells at high throughput. In our method, a microfluidic droplet generator is constructed with soft lithography using polydimethylsiloxane (PDMS) layers. Millions of 1.5 nL nanoliter monodisperse agarose-in-oil microdroplets are produced using a flow-focusing channel geometry with a high generation rate of 444 droplets per second. An individual cell along with a microbead functionalized with multiplex primers for the STR targets are statistically encapsulated within the droplets. The beads serve as amplicon-binding substrates to maintain the monoclonality of STR analysis by preventing the cross-contamination of DNA products from different droplets and different cells. The unique thermo-responsive sol-gel switching property of agarose enables the gel droplets containing the individual cells to be flexibly processed for cell lysis, amplification, mechanical manipulation and long-term storage. Following lysis and digestion of the cell-containing droplets in a chemical lysis buffer containing sodium dodecyl sulfate (SDS) and proteinase K, genomic DNA is released from the cell but remains trapped in the porous agarose network. The gel This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. 2 droplets are then equilibrated with PCR mixture and redispersed in the carrier oil by mechanical agitation to form a uniform emulsion of nanoliter reactors. Massively parallel single-cell emulsion PCR is then performed in a single PCR tube using a conventional thermocycler, during which STR loci information from an individual cell is transferred onto the microbead within a droplet. No droplet merging is observed using a silicon oil mixture containing 1% Triton X-100 with both bovine serum albumin (BSA) and Tween 80 in PCR mixture. Following PCR amplification, the beads are recovered from the droplets by removing the oil and melting the agarose to disrupt the droplets. To analyze the STR products immobilized on the beads, secondary PCR was carried out to transfer the STR information into free solutions. The beads are quantified and diluted to the stochastic limit in standard PCR tubes or 96-well plates to serve as the DNA templates for a secondary PCR amplification. Finally, the secondary-PCR products from single beads are detected using a conventional capillary electrophoresis (CE) system for fragment sizing analysis. To explore the utility of this method for forensic DNA typing, a 9-plex STR system was developed with eight core STR loci including D3S1358, D5S818, D7S820, D8S1179, D13S317, D21S11, vWA, and TH01 plus a sex marker, amelogenin. The protocols for the microbead-based multiplex PCR were initially optimized both in bulk solutions and microdroplets using standard 9947A female and 9948 male genomic DNA to validate the method. Under optimized procedures, we could obtain complete STR profiles of standard GM09947 (female) and GM09948 (male) human lymphoblast cell lines starting from the droplets containing 0.15 cells and 0.9 beads per droplet. The results indicated the conservation of single-genome integrity within droplets during cell lysis as well as the successful transformation of STR information from cells to microbeads. The mixtures of two human lymphoblast cells GM09947 and GM09948 were tested with cell ratios of 1:1 and 2:1. Although mixed STR profiles were observed when the cells stuck to each other, STR profiles from single cells were selectively detected in both cases and the number of dual profiles was reduced with lower cellular concentration as expected. The ability of our method to detect multiple STR loci from single cells in a mixture with high-throughput enables it to be applicable to analyzing evidence samples involving low-abundance materials and multiple suspects thereby solving the classic mixture analysis problem. This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.