title: Internal validation of the GlobalFiler™ Express PCR Amplification Kit for the direct amplification of reference DNA samples on a high-throughput automated workflow.
abstract: The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5ng, however, the optimum range determined was 2.5-10ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR(®) Identifiler(®) Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit.
authors.affiliation: Institute of Applied Genetics, Department of Forensics and Investigative Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA. Electronic address: shahida.flores@unthsc.edu.
authors.forename: Shahida
authors.initials: S
authors.lastname: Flores
authors.affiliation: Institute of Applied Genetics, Department of Forensics and Investigative Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.affiliation: Institute of Applied Genetics, Department of Forensics and Investigative Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: King
authors.affiliation: Institute of Applied Genetics, Department of Forensics and Investigative Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA; Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia.
authors.forename: Bruce
authors.initials: B
authors.lastname: Budowle
mesh_terms: D001331:Automation; D004247:DNA / Q000235:genetics* / Q000592:standards; D019075:Electrophoresis, Capillary; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D012015:Reference Standards; D015203:Reproducibility of Results; D013269:Stochastic Processes
chemical_list: D004247:DNA
keywords: Direct amplification; Forensic STR loci; GlobalFiler Express™; Reference DNA samples; Short tandem repeats; Y-indel