Biomedical subjects

S A Greenspoon

Publications and source records attributed to S A Greenspoon.

3 recordsLinked to original sources

PubMed2006

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.

Automation↗

PubMed2000

Validation of the PowerPlex 1.1 loci for use in human identification.

STR typing is now the favored method of DNA analysis for the purposes of human identification in the forensic community. The Forensic Services Division of the Detroit Police Department has completed its validation of the PowerPlex 1.1 loci (CSF1PO, TPOX, THO1, vWA, D16S539, D7S820, D13S317, and D5S818) for use in forensic casework. Detroit Metro Area Red Cross samples were typed from each of five racial/ethnic groups--the Hispanic, Caucasian, African American, Asian, and American Indian populations--and allele and genotype frequencies were calculated. A rare off-ladder variant (9.1 allele at D7S820) was identified among the database samples. A number of validation studies were performed. DNA was extracted from different substrates and typed as expected, except for the DNA extracted from leather (signal absent from the D16S539, D7S820, D13S317, CSF1PO, and TPOX loci) and from dirt (no PCR product generated). The minor contributor in the mixture study (250 pg input DNA) was facile to discern. The Concordance study, the variety of fluids from the same individual, and NIST standards studies all produced the expected results. Finally, STR data confirmed previous DNA typing results from adjudicated casework samples.

Body Fluids↗

PubMed1998

QIAamp spin columns as a method of DNA isolation for forensic casework.

The Detroit Police Crime Lab has historically used Chelex as a method to isolate DNA for amplification and typing of bloodstains at the HLADQA1, PM and D1S80 loci. However, preliminary validation of several STR systems for casework has demonstrated that the Chelex procedure is not the best method of DNA isolation for STR amplifications for our purposes. Long term storage at -20 degrees C in the presence of unbuffered Chelex beads (approximately 1 year), combined with multiple freeze thaws, resulted in signal loss at a locus for many database samples. Therefore, we have employed the QIAamp spin column as an alternative method of DNA isolation for amplification and typing of STR loci currently being validated for use in the laboratory. Moreover, we determined that QIAamp isolated DNA is also suitable for HLADQA1, PM and D1S80 typing. A matrix study was performed to determine if the QIAamp DNA procedure would give better results on bloodstains deposited on "problem surfaces" such as leather, dirt and various dyed fabrics. Again, QIAamp isolated DNA was more readily typeable than Chelex isolated DNA. We successfully replaced the phenol/chloroform extraction steps utilized in our laboratory for differential extractions, a commonly used method for separating sperm and non-sperm fractions of sexual assault evidence, with the QIAamp spin columns. The QIAamp extracted DNA performed as well in all PCR amplification and typing procedures tested (PM, HLADQA1, D1S80, and STR (PowerPlex)) as the phenol/chloroform Centricon isolated or EtOH precipitated DNAs. Thus we concluded that QIAamp spin columns are a superior method for isolating DNA to be typed for a variety of loci.

Alleles↗