01985nam a2200181   4500001000700000100004100007101000800048200015500056210007900211215002000290300013900310330123400449606003301683606002201716606002701738700001401765701002401779393968  a20160531d2014       y0pory50      ba  aeng  aMicrochip analyzer for forensic short tandem repeat typing of single cellsbRecurso eletrónicoefinal technical reportfTao Geng, Richard A. Mathies  a[Rockville, MD]cNational Criminal Justice Reference Service (NCJRS)d2014  a1 CD-ROMd12 cm  aResearch 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.)  aShort 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.  aIDENTIFICAÇÃO DE PESSOAS  aQUÍMICA FORENSE  aANÁLISE LABORATORIAL  aGENGbTao  aMATHIESbRichard A.