RESUMEN
UNLABELLED: Short tandem repeat (STR) DNA typing is a global standard for human identification. Current practice involves highly trained forensic analysts, operating in a laboratory setting, using multiple instruments to process samples and analyze the data. Here, we report the developmental validation of a fully integrated and automated DNA profiling system, the RapidHIT® System, capable of producing up to five high quality STR profiles with full controls in approximately 90min using PowerPlex®16 HS RapidHIT chemistry. The system integrates all sample handling steps: starting from lysis of cells on buccal swabs or other buccal sample types through DNA extraction, normalization, amplification,capillary array electrophoresis, detection, and integrated software analysis. The results describe the developmental validation of the RapidHIT™ System for buccal samples processed with the DNA IQ™ extraction chemistry using a guandinium chaotropic agent and paramagnetic beads followed by amplification using a modified version of PowerPlex 16 HS chemistry (PowerPlex 16 HS RapidHIT chemistry), and capillary electrophoresis with manual review of genotyping data following interpretation guidelines. All processing from the buccal swab to generation and processing of the profile occurs on the RapidHIT platform. RESULT: are concordant with traditional methods, with 88% first pass success rates for both the CODIS and PowerPlex 16 loci. Average peak height ratios were 0.89 for buccal swabs. The system produces full profiles from swabs with at least 176 ng of saliva DNA. Rapid DNA identification systems will significantly enhance capabilities for forensic labs, intelligence, defense, law enforcement, refugee and immigration applications, and kinship analysis.
Asunto(s)
Antropología Forense , Mucosa Bucal/metabolismo , Humanos , Repeticiones de Microsatélite/genética , Reacción en Cadena de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
We describe a programmable microfluidic system with onboard pumps and valves that has the ability to process reaction volumes in the sub-microlitre to hundred microlitre range. The flexibility of the architecture is demonstrated with a commercial molecular biology protocol for mRNA amplification, implemented without significant modification. The performance of the microchip system is compared to conventional bench processing at each stage of the multistep protocol, and DNA microarrays are used to assess the quality and performance of bench- and microchip-amplified RNA. The results show that the microchip system reactions are similar to bench control reactions at each step, and that the microchip- and bench-derived amplified RNAs are virtually indistinguishable in differential microarray analyses.