High-performance forensic DNA profiling using fluorescence energy transfer primers and a 96-lane microfabricated capillary array electrophoresis device

We present a 96-channel microfabricated capillary array electrophoresis (μCAE) device for fluorescence energytransfer (ET) dye labeled short tandem repeat (STR) genotyping. The 96-channel μCAE system produced high-speed (<30 min), highly parallel DNA separations with single-base resolution. To achieve higher sensitivity of STR typing, we adapted ET dye labeling method, and the resultant ET 16-plex amplicons which are the markers used in the commercial STR typing kit PowerPlex 16 displayed 1.6-8.7 fold increased fluorescence intensities and improved limit of detection compared to those produced using the single-dye labeled multiplex kit. In addition, the ET 16-plex generated complete STR profiles with only 28 PCR cycles; this capability should improve low copy number (LCN) typing by reducing the amplification time and background, thereby providing higher successful profiling rate. To confirm the practical advantages of ET-labeled primers, six previously problematic sexual casework samples were tested and only the ET 16-plex kit was able to capture additional DNA allele data. The successful development and demonstration of ET cassette labeled primers for higher sensitivity STR typing offers a simple solution to improving current commercial multiplex typing capability. The pairing of ET cassette technology with the μCAE system illustrates not only an enhanced STR typing platform, but also a significant step toward a higher-efficiency integrated forensic system.