Research report submitted to the U.S. Department of Justice, Award Number: 2009-DN-BX-K180. Ficheiro de 3,15 MB em formato PDF (39 p.)

Short tandem repeat (STR) typing at the single-cell level is a promising tool for human forensicidentification 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. CONTENTS: 1. Introduction. 2. Methods. 2.1 Microfluidic device fabrication. 2.2 Preparation of primer-functionalized beads. 2.3 Cell culture and sample preparation. 2.4 Microdroplet generation. 2.5 Cell lysis and DNA purification. 2.6 Emulsion PCR. 2.7 Bead recovery. 2.8 Secondary PCR and fragment sizing analysis. 3. Results. 3.1 Microbead-based solid-phase PCR in bulk solutions. 3.2 Agarose microdroplet-based emulsion PCR. 3.3 Single-molecule PCR in microdroplets. 3.4 Single-cell STR typing in microdroplets. 4. Conclusions. 5. References. 6. Dissemination of research findings.