Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.     

Developmental validation of the quantifiler real-time PCR kits for the quantification of human nuclear DNA samples

The Quantifiler Human DNA Quantification Kit and the Quantifiler Y Human Male DNA Quantification Kit were designed for the quantification of human genomic DNA in forensic samples. The kits use a real-time PCR-based process to quantify, respectively, total human DNA or human male DNA only. We report the results of a developmental validation study that we performed with the Quantifiler Kits, following the official SWGDAM guidelines. The Quantifiler Kits were tested for performance criteria such as species specificity, sensitivity, stability, precision and accuracy, and in addition, were tested with forensic case-type samples and mixed (male:female) DNA samples. The Quantifiler Kit methods were highly specific for human DNA, and could detect as little as 32 picograms of DNA using 2 microL of sample per assay. The accuracy and precision of the Quantifiler Kit methods was comparable or superior to that of other quantification methods.