Forensic analysis of deoxyribonucleic acid (DNA) represents an important facet of criminal investigations. DNA extraction, the first step in sample processing where DNA is released from biological samples and isolated, is crucial for subsequent analysis. Minimizing loss of DNA during extraction as well as ensuring complete lysis of DNA-containing cells are two factors that must be considered when choosing an extraction method. Extraction procedures that minimize sample transfers, specifically single-tube extractions, are ideal for minimizing DNA loss. A DNA extraction kit made by ZyGEM (Hamilton, New Zealand) incorporates the use of a recently characterized proteinase; EA-1 achieves a single-tube extraction that requires no further purification. However, research published on this method reported that the extraction was unable to release DNA from sperm cells. Sexual assault cases routinely require DNA to be extracted from sperm cells, thus the ZyGEM method would be unsuitable for treatment of such samples. It could, however, possibly be used as part of a differential extraction procedure for samples containing a mixture of epithelial cells and sperm cells. The initial step in a differential extraction is the preferential lysis of any non-sperm cells. The DNA from these cells is separated and removed from the intact sperm, which are then lysed using more robust methods. The current research discusses the process of differential extraction and the investigation of an alternative method for sperm cell lysis. Development of this method was aided by studying the physical structure of sperm cells. Sperm DNA is packaged by small proteins, the protamines that replace histones during spermiogenesis. These proteins are comprised primarily of the amino acids Arginine and Lysine. The serine proteinase Trypsin, which cleaves peptides at Arginine and Lysine, was investigated as an alternative enzyme for sperm lysis in a differential extraction procedure. A sperm cell extraction method with Trypsin was developed, using the forensicGEM extraction to purify the sample following lysis with Trypsin. This method was compared to an extraction of sperm with Qiagen QIAamp® DNA Investigator kit. The results show that the new method using Trypsin and ZyGEM yields between 4 and 12 times more DNA than the Qiagen method. When low template samples were extracted and amplified, the new method was able to generate a full profile while samples extracted with Qiagen lost over 80% of the profile due to low yield in the extraction. Analysis of Peak Height (PH) showed the new method had slightly lower peak heights compared to Qiagen when similar amounts of DNA were amplified, however Peak Height Ratio (PHR) was not reduced in the new method. The results indicate that a new extraction method using Trypsin and ZyGEM provides greater amounts of DNA from extractions of sperm cells and that the method should be further developed into a differential extraction protocol to aid in mixture sample processing. / 2022-03-31

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

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.

There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures. Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile. Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.

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.

Differential extraction has proven to be a challenging and time-consuming process, often requiring up to six hours of a forensic analyst’s concentration. With the ever-increasing backlog of sexual assault evidence kits, the forensic community is using new ways to diminish this backlog, including more streamlined evidence processing and sample analysis. The goals for processing sexual assault samples include efficient recovery of sperm deoxyribose nucleic acid (DNA), simplified sample processing, and the development of a profile eligible for forensic analysis. Cost and time can also be limiting factors. The Cotton Research Lab at Boston University has developed a novel method of differential extraction that combines separation of epithelial and sperm cell fractions, nuclease treatment to reduce female DNA carryover and a direct-cell lysis protocol. With the exception of a single centrifugation step, the entire protocol is conducted using a thermalcycler in the DNA extraction laboratory. Thus, the process is a Temperature Controlled Differential Extraction (TCDE), and has been effectively adapted for use with liquid, dried, and aged samples. The purpose of this research is to explore methods which further adapt the protocol for best use with forensic casework samples, namely vaginal swabs. Sexual assault evidence collection kits may contain a variety of items, and commonly include cotton swabs for the collection of fluids from intimate sources. To simulate casework-type samples, swabs were prepared with liquid epithelial cell preparations and various semen dilutions (ranging from 1:1 to 1:1000). Amendments were made to the TCDE protocol for best DNA recovery from a swab, and buffer changes were made to enhance compatibility with polymerase chain reaction (PCR)-amplification kits widely utilized in forensic labs. Finally, post-coital swabs from female donors were analyzed using the TCDE protocol with modifications for forensic casework samples. Preliminary testing of casework-type swabs with protocol modifications showed high yields of DNA and successful separation of epithelial and spermatozoa fractions. The epithelial fraction, when yielding a mixed profile, demonstrated a clear major female contributor, and the spermatozoa fractions showed little to no female carryover, often exhibiting single source male profiles. The TCDE protocol with modifications for casework-type samples requires approximately 2 hours and 30 minutes of an analyst’s time, from the moment the swab is removed from its evidence packaging to an extraction ready for DNA quant and short tandem repeat (STR) amplification. The method provides increased DNA recovery, can be used with various amplification kits, and generate probative profiles and is time efficient. This robust and promising new method that has the potential to be automated and to contribute to the effort to reduce the backlog in the analysis of sexual assault evidence kits.

The separation of sperm from female epithelial cells has been a topic of interest in forensic DNA (deoxyribonucleic acid) analysis since the origin of the field. One of the most needed applications of DNA analysis in the identification of the perpetrator of a sexual assault, as often there is little to no other evidence for identification. The largest hurdle to forensic DNA analysis in these cases is that vaginal or oral swabs from sexual assaults will have a mixture of the victim’s epithelial cells and the perpetrator’s sperm cells. It is well known that the analysis of complex mixtures can be difficult to impossible, especially when there is an added concern of low template DNA. Separating these cell types in the mixture evidence is the best way to avoid the need to deduce these difficult mixtures. Sperm and Epithelial Cells are morphologically different both in cell shape and DNA packaging. Nuclear DNA in epithelial cells are more loosely packaged around histones in a structure called a nucleosome. Sperm DNA is tightly packaged around protamines rather than histones. These DNA packaging differences can be utilized to preferentially lyse sperm and epithelial cells in order to separate them. Traditionally this is done by lysing epithelial cells with sodium dodecyl sulfate (SDS) and proteinase K (PK), separating this epithelial DNA from the sperm by centrifugations and finally lysis of the sperm using dithiothreitol (DTT) which reduces the disulfide bonds in the sperm DNA packaging. This method was developed by Peter Gill in 1985 and is still used by forensic laboratories to date. This differential extraction is very labor intensive and time consuming. This dual-enzyme differential extraction can be performed in roughly one hour, which is highly advantageous with the large amount of backlogged sexual assault cases that forensic laboratories have. This work was undertaken to improve the separation of epithelial DNA from sperm cells in the dual-enzyme differential extraction. Here we found that the DNA carryover into the sperm fraction was due to a combination of an inability to completely separate the non-sperm fraction liquid from the sperm pellet and the decreased efficiency of ZyGEM to fully lyse epithelial cells in clumps. The solution to this problem includes the addition of a wash of the sperm pellet after initial separation of the fractions. This wash step decreased the concentration of epithelial DNA to the point that its detection may only occur with very low concentrations of sperm DNA. / 2017-11-03T00:00:00Z

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.

A two-step protocol has been devised as a rapid and selective alternative to conventional differential extraction techniques with an increased recovery of DNA. The protocol involves pressure cycling with the Barocycler® NEP 2320 from Pressure Biosciences. Inc. in alkaline conditions for epithelial cell lysis and removal. This step is followed by alkaline lysis at 95º C for extraction of sperm cell DNA. At 1:1 or 2:1 female to male cell ratios, high selectivity and complete separation can be achieved. But at higher ratios, male allelic dropout is observed. This protocol has been modified to generate a clean male profile at a 20:1 cell ratio through optimization of NaOH concentration and inclusion of an additional pressure cycling step. Validation studies have been performed to assess the efficiency of this method under various conditions. An additional immunomagnetic cell capture pretreatment allowed for nearly complete separation at cell ratios of up to 200:1.