The sexual assault kit backlog in the United States has become an increasing problem over the years. Combined with the number of kits laboratories receive with how it takes to extract the deoxyribonucleic acid (DNA) from the cells, it is hard for labs to keep up with the demand. The extraction method used is called differential extraction, where the epithelial cells from the victim are separated from the sperm cells from the perpetrator into different fractions. The Temperature Controlled Differential Extraction (TCDE) method is a novel procedure developed by the Cotton Lab at the Boston University School of Medicine and designed to decrease the extraction time while performing just as well, if not better, than traditional differential extraction methods. The TCDE method uses a series of temperature-controlled enzymes to lyse cells and purify the DNA extract. The purpose of this study is to compare this TCDE method to a method implemented by QIAGEN using the EZ1® Advanced biorobot for purification, which is used in many forensic laboratories. Ten female donors each received ten cotton swabs for vaginal cell collection; cotton swabs are typically found in sexual assault kits. Each swab then received either 5ng, 25ng, or 50ng of male DNA in the form of sperm cells. One half of the swab was processed using the TCDE procedure while the other half was processed using the EZ1® method. The TCDE method results in three fractions: the Epithelial Fraction (EF), the Material Fraction (MF), and the Sperm Fraction (SF). The EZ1® protocol was modified to include the additional MF. Results of both the quantitation data as well as the electropherograms (EPGs) produced are compared between the two methods. The quantitation data for the EF shows a variable amount of female DNA recovered due to the uncontrolled amount of female epithelial cells added to the swabs from the donors. The MF shows that large amounts of female epithelial DNA remain in the fraction for the EZ1® protocol and not the TCDE protocol because of the nuclease activity of one of the enzymes. The remaining male DNA on the MF can be used to compare to a known male profile, showing that there is valuable data potentially left behind. Regarding the SF, the EZ1® protocol resulted in a higher yield of DNA than the TCDE, however, the TCDE SF electropherograms are still able to be used for comparisons against known male profiles. The TCDE protocol cuts extraction time by almost half, and the quantitation results and EPGs prove that this method has the potential to become the new standard method of differential extraction.

Short Tandem Repeat (STR) analysis has become a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. This report describes the use of laser microdissection (LMD) for the separation of pure populations of spermatozoa from two-donor cell mixtures. In this study, cell separation was demonstrated by microscopic identification of histologically stained spermatozoa and female buccal cell mixtures, and STR analysis of DNA obtained from the separated sperm cells. Clear profiles of the male donor were obtained with the absence of any additional alleles from the female donor. Five histological stains were evaluated for use with LMD and DNA analysis: hematoxylin/eosin, nuclear fast red/picroindigocarmine, methyl green, Wright's stain, and acridine orange. Hematoxylin/eosin out-performed all other stains however nuclear fast red/picroindigocarmine could be used satisfactorily with STR analysis. In addition, three DNA isolation methods were evaluated for LMD collected cells: QIAamp (Qiagen), microLYSIS (Microzone Ltd.) and Lyse-N-Go (Pierce Chemical Co.). MicroLYSIS performed poorly, yielding low levels of PCR product. Lyse-N-Go extraction was effective for the recovery of DNA from LMD collected sperm cells while QIAamp isolation performed best for the recovery of DNA from LMD collected epithelial cells. LMD is shown to be an effective, low-manipulation separation method that enables the recovery of sperm while excluding epithelial cell DNA.

It is common practice for DNA STR profiles to be analyzed using an analytical threshold (AT), but as more low template DNA (LT-DNA) samples are tested it has become evident that these thresholds do not adequately separate signal from noise. In order to confidently examine LT-DNA samples, the behavior and characteristics of the background noise of STR profiles must be better understood. Thus, the background noise of single source LT-DNA STR profiles were examined to characterize the noise distribution and determine how it changes with DNA template mass and injection time. Current noise models typically assume the noise is independent of fragment size but, given the tendency of the baseline noise to increase with template amount, it is important to establish whether the baseline noise is randomly found throughout the capillary electrophoresis (CE) run or whether it is situated in specific regions of the electropherogram. While it has been shown that the baseline noise of negative samples does not behave similarly to the baseline noise of profiles generated using optimal levels of DNA, the ATs determined using negative samples have shown to be similar to those developed with near-zero, low template mass samples. The distinction between low-template samples, where the noise is consistent regardless of target mass, and standard samples could be made at approximately 0.063 ng for samples amplified using the Identifiler^TM Plus amplification kit (29 cycle protocol), and injected for 5 and 10 seconds. At amplification target masses greater than 0.063 ng, the average noise peak height increased and began to plateau between 0.5 and 1.0 ng for samples injected for 5 and 10 seconds. To examine the time dependent nature of the baseline noise, the baselines of over 400 profiles were combined onto one axis for each target mass and each injection time. Areas of reproducibly higher noise peak heights were identified as areas of potential non-specific amplified product. When the samples were injected for five seconds, the baseline noise did not appear to be time dependent. However, when the samples were injected for either 10 or 20 seconds, there were three areas that exhibited an increase in noise; these areas were identified at 118 bases in green, 231 bases in yellow, and 106 bases in red. If a probabilistic analysis or AT is to be employed for DNA interpretation, consideration must be given as to how the validation or calibration samples are prepared. Ideally the validation data should include all the variation seen within typical samples. To this end, a study was performed to examine possible sources of variation in the baseline noise within the electropherogram. Specifically, three samples were prepared at seven target masses using four different kit lots, four capillary lots, in four amplification batches or four injection batches. The distribution of the noise peak heights in the blue and green channels for samples with variable capillary lots, amplifications, and injections were similar, but the distribution of the noise heights for samples with variable kit lots was shifted. This shift in the distribution of the samples with variable kit lots was due to the average peak height of the individual kit lots varying by approximately two. The yellow and red channels showed a general agreement between the distributions of the samples run with variable kit lots, amplifications, and injections, but the samples run with various capillary lots had a distribution shifted to the left. When the distribution of the noise height for each capillary was examined, the average peak height variation was less than two RFU between capillary lots. Use of a probabilistic method requires an accurate description of the distribution of the baseline noise. Three distributions were tested: Gaussian, log-normal, and Poisson. The Poisson distribution did not approximate the noise distributions well. The log-normal distribution was a better approximation than the Gaussian resulting in a smaller sum of the residuals squared. It was also shown that the distributions impacted the probability that a peak was noise; though how significant of an impact this difference makes on the final probability of an entire STR profile was not determined and may be of interest for future studies.

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.

Criminology is a science dealing with the protection of citizens and state from infringement. Criminology uses mostly biological or genetic methods for crime detection. Forensic traces which are collected by forensic experts on the scene are the key items of those methods. Forensic genetics is among the most important forensic subdisciplines. Forensic genetics uses DNA analysis for identification. The main aims of this study are description and importance of biological, anthropological and genetic methods in criminology, different ways of forensic identification, division and collection of forensic traces, characterization and course of forensic DNA analysis and DNA profiling. Key words Criminology, forensic methods, forensic identification, forensic trace, forensic biology, anthropology and genetics, information systems, forensic DNA analysis, DNA profile.