Forensic analysis of DNA from sexual assault kits is a laborious process. These samples may be a mixture of sperm and male or female epithelial cells (E-cells). Generally, it is the sperm cells that are of greatest forensic value. Since its introduction in 1985 by Gill, Jefferys and Warrett, differential extraction has remained an essential pre-PCR extraction procedure adopted by most forensic laboratories for the preferential lysis of E-cells and isolation of sperm cells/male fraction prior to DNA profiling. The differential extraction procedure operates based on the packaging of DNA in these two types of cells. The E cells are first lysed by sodium dodecyl sulfate (SDS) and Proteinase K which leaves the sperm cells intact. The mixture is centrifuged leaving E-cell DNA in the supernatant and sperm cells in the pellet. After several wash steps to remove residual E cell DNA, the sperm fraction is then subjected to lysis using SDS, proteinase K, and dithiothreitol (DTT). DTT reduces the disulfide bonds present in the sperm nucleus, thereby releasing sperm cell DNA. The traditional Gill method of differential extraction, while proven to be highly effective in providing two separate fractions for a simplified interpretation of profiles, is a labor intensive and time-consuming process, requiring approximately six hours of an analyst’s concentration. In a casework scenario where an evidence sample is of a higher E cell concentration compared to sperm cells, it is inevitable to obtain mixture profiles that becomes more difficult to interpret. To mitigate carryover from the female fraction, the sperm cell fraction is usually subjected to multiple wash steps. Furthermore, the resulting fractions must be subjected to additional pre-PCR DNA purification procedures to remove PCR inhibitors such as SDS and Proteinase K which result in varying degrees on DNA loss. Progress has been made over the years to introduce methods that allow for PCR-ready lysates without additional purification steps, often referred to as direct lysis methods. However, none have been proven to be viable options for use in sexual assault samples. Our laboratory has developed a novel differential extraction procedure that is not only time-efficient and less laborious but also utilizes a direct-lysis procedure requiring no further pre-PCR purification for most samples. The novel procedure uses ZyGEM, which contains the thermophilic EA1 protease proven to effectively digest biological samples and produce PCR-ready lysates suitable for downstream nucleic acid amplification, thereby minimizing DNA loss. The procedure uses a multi-enzymatic approach and utilizes the different optimal activity temperatures of the enzymes to perform most of the process in a DNA extraction lab thermocycler, requiring only a single centrifugation for the usual separation of the E-cell fraction and no subsequent washing steps for the sperm cell fraction. It has the potential to be a rapid, robust procedure that can be easily implemented in any forensic laboratory. This thesis will describe the procedure and report progress in the procedure optimization. / 2019-10-24T00:00:00Z

In forensic casework, recovery of more deoxyribonucleic acid (DNA) generally leads to a better chance of obtaining a robust and reliable DNA profile. However, DNA evidence often contains a very low amount of cells, therefore, the importance of proper collection and storage to protect the DNA and ensure that maximum collection of cells is achieved cannot be over emphasized. New techniques and inventions have made the collection of DNA evidence more efficient and consistent through the development of different types of swabs, lysing buffers and various other improvements. Even with the development of these improvements, the ability to maximize the collection of cellular material from a substrate is still impeded by various issues in the extraction process along with the structural properties of swabs used for collection. Research by Adamowicz et al. found that when extracting buccal and blood cell samples collected on cotton swabs, using the recommended protocol for swabs with the QIAamp DNA Investigator extraction kit, over 50% of the recoverable DNA is retained on the swab or lost through the extraction process [1]. Although cotton swabs are very good at absorbing biological material, they exhibit low efficiency of DNA sample release. Additional DNA may be lost during the extraction process. An optimal method of collection and extraction for forensic samples will maximize the collection and release of cellular material and minimize the loss of cellular DNA in the extraction process. The design of the Mawi DNA Technologies’ iSWABTM collection device allows for the release of cells captured from any type of swab into a proprietary lysis and stabilizing iSWABTM buffer. The combination of the mechanistic release of cells and the proprietary lysis buffer claims to maximize the collection of cells from single or several swabs in a pre-measured amount of buffer while eliminating the potential for bacterial growth and contamination. The iSWABTM Device is designed with three prongs and contains cell lysis buffer with DNA stabilization chemistry. As the swab is taken out of the collection device, the prongs provide resistance and essentially squeeze the excess solution and cells off of the swab. Following collection of the cellular material, cell lysis is achieved by incubation in the lysis buffer for 3 hours at room temperature. No additional reagents are necessary. This study investigated whether the Mawi DNA Technologies’ iSWABTM collection device and buffer could be considered as an alternative method to maximize the recovery of cells/DNA from swabs. Experiments were conducted to test the efficiency and forensic application of the device. The following parameters of the iSWABTM buffer and collection device were tested: 1) ability to collect dried stains; 2) ability to recover cellular material from different types and conditions of swabs; 3) ability to lyse different cell types; 4) ability to stabilize DNA over an extended period of time; and, 5) ability to perform in downstream Polymerase Chain Reaction (PCR) testing and produce quality STR profiles. Cumulatively, the data indicates that the iSWABTM-ID collection device is simple, fast and convenient while providing high DNA recovery. Some modifications or additional procedure developments can be done to facilitate the application for use with samples containing very small amounts of biological materials.

DNA (Deoxyribonucleic Acid) must be extracted from the cell before it can be amplified using the PCR (Polymerase Chain Reaction) to generate an STR (Short Tandem Repeat) profile. Although extraction methods such as binding DNA to silica remove PCR inhibitors, they have complex procedures that can lead to partial DNA loss. Evaluating the efficiency of an extraction method can be difficult, as the original amount of input DNA may be unknown. Knowledge of the amount of DNA expected to be present in a sample can provide quantitative information on an extraction method’s efficiency, as the recovery of DNA from a DNA extraction process can be calculated using the expected DNA value. Theoretically, while using a direct lysis extraction method, all the cells in the sample are lysed and present in that single tube, so quantitative PCR (qPCR) values of samples extracted using a direct lysis method such as forensicGEMTM can be used as an expected yield value. Validation studies determine if a method is reliable, reproducible, and robust. An internal validation study, following the Federal Bureau of Investigation’s Quality Assurance Standards (QAS) for Forensic DNA Analysis Methods, was performed for the Maxwell® FSC instrument, a robotic DNA extraction instrument designed to remove PCR inhibitors from forensic samples. Results from this study were compared to results obtained on a previously validated Maxwell® 16 instrument. The validation study was successful, as the study demonstrated the instrument could process known/non-probative evidentiary samples, is sensitive, precise, accurate, can process mixture samples, and had no detectable contamination in the process. An added study, done post validation, compared the amount of DNA obtained using a “direct lysis method” with the amount of DNA obtained using samples equivalent to those used in the instrument validation. However, upon comparing the amount of DNA recovered using the Maxwell® FSC instrument to the amount of DNA obtained using a direct lysis method, which in theory would recover 100% of the DNA, the percent yield for serially diluted blood samples was low. Only 10.79% of the DNA was recovered for the 1:10 dilution, 14.44% was recovered for the 1:100 dilution, and 8.00% was recovered for the 1:1000 dilution. The DNA IQTM System – Small Sample Casework kit uses the same chemistry and the same reagents as the Maxwell® FSC DNA IQTM Casework Kit. To provide additional data on DNA recovery, the Maxwell® FSC Study was replicated using the DNA IQTM System – Small Sample Casework Kit, following the manufacturer’s instructions for manual extractions. The study showed that about 77.71% of the DNA is lost when using neat samples, 96.88% is lost for the 1:10 dilution samples, 95.22% is lost for 1:100, and 99.00% for 1:1000. Steps from the DNA IQTM System – Small Sample Casework Protocol were identified where, potentially, the DNA loss could have occurred. These included DNA left in the waste products, DNA left on the swatch, and DNA left on the Resin after elution. Study data identify experimentally two major sources of DNA loss: the waste and the swatch. When quantifying the “waste,” for almost every individual sample, more DNA remained in the waste than was found in the initial extract using the DNA IQTM System – Small Sample Casework Kit. The high amounts of DNA in the waste indicates that the DNA IQ™ Resin does not bind to all the available nucleic acid in the solution. The data and other data characterizing DNA loss at particular steps of the protocol are described in this study. Modifications to the protocol are described, which were made, in an attempt, to increase the amount of DNA bound to the Resin that could subsequently be recovered. Laboratories that use DNA IQTM reagents need to be aware that a significant amount of the sample may be lost when following the procedure. Preliminary results indicate that yield can be improved by modifying the procedure to either increase the Resin volume, increase the incubation temperature, or recapture DNA from the first waste.

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