High-performance integrated genetic analyzers for forensic DNA typing.

摘要

Microfabrication technology offers great potential for the integration of all steps of forensic DNA typing onto a single microdevice. This integration should enable rapid, low-cost and reliable short tandem repeat (STR) analysis not only in forensic laboratories but also at crime scenes or other relevant point-of-analysis. As a first step towards making on-site STR typing possible, I developed a microdevice consisting of a 160-nL polymerase chain reaction (PCR) chamber and a 7-cm capillary electrophoresis (CE) channel and a portable instrument for its operation. A four-plex mini Y STR typing system was constructed for testing the capability of this microsystem for forensic STR typing. The successful analyses of casework and mixture samples validate the concept of forensic STR typing on a portable microfluidic system.To critically evaluate the capabilities of this portable system and the feasibility of DNA typing at a crime scene, real-time DNA analyses using a 9-plex autosomal STR typing system on a modified PCR-CE microdevice containing a co-injection structure for fragment sizing calculation were carried out at a mock crime scene. Blood stain collection, DNA extraction, STR analysis on the PCR-CE microsystem, and a DNA profile search against a mock CODIS database were successfully conducted within 6 hours of crime scene arrival. This demonstration establishes the feasibility of real-time DNA typing at a crime scene or other point-of-care situations.Finally, to achieve a total integrated analysis system for real-time STR typing, an up-front sequence-specific DNA extraction and concentration method using magnetic beads was developed and incorporated into the PCR-CE microdevice. Fragmented genomic DNA was hybridized with capture probes and immobilized onto magnetic beads via streptavidin-biotin binding in microchannels. The bead-DNA conjugates were then transported to a PCR reactor for amplification followed by inline injection using a novel capture concentration method for CE separation. This fully integrated system significantly advances the forensic DNA typing by providing a high-performance platform with sample-in-answer-out capability for real-time human identification.