Rapid PCR protocols for forensic DNA typing on six thermal cycling platforms.

Rapid PCR protocols for the amplification of typing STR multiplexes were evaluated on six different thermal cyclers. Through the use of a faster DNA polymerase coupled with the use of rapid thermal cyclers the amplification cycling times were reduced down to as little as 14 min using PCR primers from the commercially available multiplex STR typing kit Identifiler. Previously described two-step and three-step thermal cycling protocols were evaluated for the six thermal cyclers on 95 unique single-source DNA extracts. CE characterization of the PCR products indicates good peak balance between loci (median values greater than 0.84), and N minus four stutter ratios on averages were 30 to 40% higher than for standard Identifiler PCR conditions. Nonspecific amplification artifacts were observed, but were not observed to migrate within the allele calling bins. With the exception of one locus (D18S51) in a single sample, genotyping results were concordant with manufacturer's recommended amplification conditions utilizing standard thermal cycling procedures. Assay conditions were robust enough to routinely amplify 250 to 500 pg of template DNA. This work describes the protocols for the rapid PCR amplification of STR multiplexes on various PCR thermal cyclers with the future intent to support validation for typing single-source samples in a database laboratory.

DNA purification from crude samples for human identification using gradient elution isotachophoresis.

Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure-driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On-line DNA quantification using laser-induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification.

Developmental validation of a fully integrated sample-to-profile rapid human identification system for processing single-source reference buccal samples.

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.

Developmental validation of the PowerPlex® 18D System, a rapid STR multiplex for analysis of reference samples.

As short tandem repeat markers remain the foundation of human identification throughout the world, new STR multiplexes require rigorous testing to ensure the assays are sufficiently robust and reliable for genotyping purposes. The PowerPlex(®) 18D System was created for the direct amplification of buccal and blood samples from FTA(®) storage cards and reliably accommodates other sample materials. The PowerPlex(®) 18D System allows simultaneous amplification of the 13 CODIS loci and amelogenin along with four additional loci: Penta E, Penta D, D2S1338, and D19S433. To demonstrate suitability for human identification testing, the PowerPlex(®) 18D System was tested for sensitivity, concordance, inhibitor tolerance, and performance with thermal cycling and reaction condition variation following SWGDAM developmental validation guidelines. Given these results, PowerPlex(®) 18D System can confidently be used for forensic and human identification testing.