Despite the many advancements to forensic DNA analysis adopted by crime laboratories across the country, the most common method for the differential extraction of sexual assault samples has remained relatively unchanged since forensic deoxyribonucleic acid (DNA) typing was discovered in 1985. As the quantity and quality of extracted DNA has significant implications on the success of subsequent analysis methods, the development and optimization of effective extraction procedures is vital to progressing the field of forensic DNA analysis. The graduate students and faculty at the Boston University School of Medicine have been developing a differential extraction process that utilizes a multi-enzymatic approach to preferentially lyse and wash the cell types within temperature controlled environments. The overall procedure is less labor-intensive and time-consuming than the conventional method. Through the extraction process, the inhibitory nature of each enzyme on the amplification process is avoided, circumventing the need for an additional purification step. A single centrifugation step is required in order to pellet the sperm while the cumbersome wash steps are replaced with selective digestion in order to remove the residual epithelial cell DNA from the sperm fraction. The three enzyme used (EA1, Benzonase®, and Acrosolv) operate optimally at distinct temperatures which allows for controlled and sequential activation to achieve desired lysis and digestion outcomes. The enzymatic reactions are conducted within a DNA extraction lab thermal cycler to obtain rapid and accurate temperature changes. This novel temperature controlled differential extraction protocol has been developed and optimized for extraction of primarily liquid mixed samples in 0.2 milliliter (mL) tubes. The epithelial cell lysis and sperm cell lysis stages of the extraction contained a final reaction volume of 100 microliters (µL). Slight modifications to this 100 direct-lysis differential extraction method resulted in a similarly efficient method with a high male DNA yield (74-100%) and minimal female carryover among varying ratios of epithelial cells to sperm cells. This sensitive technique provided nearly complete profiles (14/16 loci) of the male contributor in mixed samples containing ~15,200 female epithelial cells and ~500 sperm, with complete profiles observed in mixed samples containing ~1000 sperm. This modified extraction protocol better accommodates sample sizes that may be encountered in forensic casework testing while providing a more concentrated sperm fraction, possibly eliminating the need for an additional concentration step in some dilute samples. The ease of implementation and the rapid processing time of 2-3 hours make it a great candidate for use in forensic DNA laboratories and may help alleviate backlogs of sexual assault kit. However, further work is needed to alter the composition of the sperm lysis buffer to make it compatible with currently used amplification kits. Until such time, caution must be taken in the kit selection used for amplification of extracts produced with this method. This study also demonstrated a sensitivity of the GlobalFiler® PCR Amplification Kit to inhibition by the buffers used in this extraction protocol, particularly the Orange+ Buffer. This inhibition has dramatic effects on the profile quality of the amplified sperm fractions, with extensive allelic drop-out observed even when the Orange+ Buffer concentration was scaled from 1.0X to 0.2X. Amplification using the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit showed marginal recovery in the profile quality. Other expanded-loci STR amplification kits may also demonstrate resistance to this inhibition.

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.

The typical differential extraction procedure utilized by the forensic science community to extract male deoxyribonucleic acid (DNA) from the sperm cells of the perpetrator separately from female DNA from the epithelial cells of the victim is both time-consuming and labor-intensive. This has contributed greatly to the backlog of unanalyzed sexual assault evidence collection kits (SAECK) seen in many laboratories today and has encouraged research in new methods that are more efficient and more effective in achieving better sperm DNA recovery. The Cotton Lab has developed a Temperature-Controlled Differential Extraction (TCDE) procedure geared towards attaining better sperm recovery and better distribution of male DNA in the sperm fraction (SF) to generate a single source or distinguishable male profile. The TCDE protocol is a direct-lysis procedure that utilizes highly temperature-controlled enzymes, or enzymes that are active at or near their optimal temperatures. This procedure has been previously shown to decrease extraction time significantly and to extract samples that are suitable for downstream analysis. This research specifically attempted to modify the TCDE procedure in the hopes of obtaining higher sperm DNA recovery and eliminating previous concerns of too much sperm being retained by the cotton swab material. It also compared a slightly modified TCDE procedure where the material fraction (MF) and SF are kept as separate fractions (the Separate Method) and a method that results in a recombined MF and SF (Recombined Method) to see if there was a greater distribution of the total male DNA eluted into the SF. Preliminary experimentation with swabs prepared with semen was performed to help make effective modifications. Then, vaginal swabs from eight different female donors were prepared with semen to mimic forensic casework samples and extracted using the Separate and Recombined Methods for comparison of the two extraction methods. Despite unusual epithelial cell lysis results for some samples, the quantitation of the fractions by quantitative polymerase chain reaction (qPCR) showed that for approximately half of the samples extracted using the Separate Method, a majority of total male DNA was eluted into the SF. For these samples, a single source or distinguishable male profile can be generated. However, it was also demonstrated that even with good separation, a very small proportion of the female DNA in the SF still overwhelms the male DNA that is present in much smaller amounts, particularly for the Recombined Method where there are only two fractions. Though further experimentation is necessary, these modifications proved effective in achieving high sperm recovery in the SF and generating a distinguishable male profile when extracting samples using the Separate Method. This research has confirmed that the TCDE procedure can be faster and less labor intensive while still producing clean DNA profiles in downstream analysis, and thus has the potential to be implemented in forensic laboratories after some of the concerns are addressed.