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Student Theses John Jay College of Criminal Justice

Summer 8-2020

Developing a Maceration Method for Isolation of DNA from Cancellous Bones

Bryan Bernal CUNY John Jay College, [email protected]

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Developing a Maceration Method for Isolation of DNA from Cancellous Bones

A Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science in Forensic Science

John Jay College of Criminal Justice The City University of New York

Bryan Bernal August 2020

Developing a Maceration Method for Isolation of DNA from Cancellous

Bones

Bryan Bernal

This thesis has been presented to and accepted by the office of Graduate Studies, John Jay College of Criminal Justice in partial fulfillment of the requirements for the degree of Master of Science in Forensic Science.

Thesis Committee:

Thesis Advisor: Dr. Richard Li

Second Reader: Dr. Artem Domashevskiy

Third Reader: Mr. Patrick David Carney

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Abstract

Prior to isolating DNA, it is necessary to remove any biological materials that may be present on commingled remains. Manual maceration of the outer surface is time-consuming.

This study will address this issue by developing a simple method for processing bone samples prior to DNA isolation. A liquid-based technique was applied to macerate bone fragments by incubating bone fragments in an enzyme solution, removing potential contaminants, followed by a bone tissue disruption. Swine (Sus scrofa) bones were used to simulate human bones.

Microscopic analysis suggested this method was effective for surface material removal. The methods effect on the quantities of DNA isolated was studied using a single-factor ANOVA; no significant differences in yields were observed. Direct sequencing of amplified fragments at the

Sus scrofa mitochondrial cytb locus was carried out. Electropherograms of the untreated control and treated samples were compared. No adverse effects were observed in the quality of treated samples. The method was applied to samples from six different species to test the methods potential application to animal identification. Resulting sequences were compared to a basic local alignment search tool (BLAST), and all species were correctly identified. The proposed method’s viability for application to human bone processing was studied using human bones.

DNA profiles were successfully detected using hypervariable region I in mitochondrial DNA.

This study demonstrated that the proposed processing method for bone fragments can be potentially useful for bone evidence, particularly cancellous bones, prior to DNA isolation.

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Acknowledgements

First and foremost, I would like to thank my thesis advisor, Dr. Richard Li. Over the last six years, his continued guidance, patience, commitment, and unyielding support has made this work, and my masters degree, a reality. I would like to thank my thesis committee, Dr. Artem

Domashevskiy and Mr. Patrick Carney, for all the advice and guidance they provided. I wish to thank my fellow masters’ student and wonderful friend, Jordana Fox for her collaboration work on Table 2. A big thank you to Caitlin Izzo, whose patience and willingness to help is only rivaled by her incredible kindness. To my friends and family whose constant, but unnecessary, reminders about this project have helped keep me feeling focused and positive these last few months. Finally, to Mrs. Dolores: a promise is a promise.

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Table of Contents Acknowledgements ...... ii Introduction ...... 1 Nuclear DNA and Mitochondrial DNA ...... 1 Applications for DNA Analysis of Bone ...... 2 Forensic DNA Testing of Bone ...... 6 Methods for Processing Bone ...... 10 Focus and Goals ...... 16 Materials and Methods ...... 19 Sample Procurement ...... 19 Bone Sample Maceration ...... 21 Bone Tissue Disruption ...... 21 DNA Extraction and Quantification ...... 21 Species Identification ...... 23 Human mtDNA Sequencing...... 25 Results and Discussion ...... 27 Macroscopic and Microscopic Effects of Specimen Maceration ...... 27 Effects of Specimen Maceration on DNA Yields ...... 33 Effects of Specimen Maceration on DNA Quality...... 34 In-Tube Bone Tissue Disruption ...... 39 Animal Species Identification ...... 43 Human Samples...... 45 Conclusion ...... 50

1

Introduction

Nuclear DNA and Mitochondrial DNA

Deoxyribonucleic acid (DNA), the genetic blueprint for all life, stores vast amounts of information in all nucleated cells in the human body. DNA uses the information contained in its sequence for replicating cells, creating proteins, and passing down genetic traits to future generations (Butler, 2011). In the early 1980s, DNA was first used as a means to identify an individual. Sir Alec Jeffreys, an English geneticist, was researching the human myoglobin gene when he discovered that the same areas of tandem repeat DNA (short, repetitive nucleotide sequences) were present in all human beings, and that the number of times the sequences repeated varied between individuals. Jeffreys examined X-ray blots of tandem repeat DNA and observed patterns that had noticeable similarities and differences between them. Using the data he collected, Jeffreys was able to identify individuals through their genetic code. This later became known as DNA fingerprinting, or DNA profiling (Gill, Jeffreys, & Werrett, 1985).

DNA profiling has become a familiar technique that was strengthened with the development of polymerase chain reaction (PCR). This method, which was developed around the same time as Jeffreys’, recreates the body’s natural process of DNA replication (Fisher,

Tilstone, & Woytowicz, 2009). Research and development into DNA profiling has led to the use of other repeating sections of DNA, known as short tandem repeats (STRs). These repeating portions of DNA can occur anywhere in the human genome, including areas not known to code for proteins. Within those non-coding regions, the number of repeating units of DNA will vary between individuals based on what was inherited by both parents. It is these differences that can be used to identify an individual (Schneider, 1997). 2

Lineage markers can also be used for identification. Unlike STRs, which are an inherited combination of both parents’ DNA, lineage markers are passed down as a block, or haplotype, to each generation by one parent without changing. Paternal lineage is followed using Y- chromosome markers and maternal lineage is traced with mitochondrial DNA (mtDNA) (Butler,

2011). The profiles resulting from mtDNA and Y-chromosome testing will only be able to provide circumstantial identifications because the DNA haplotype will be the same for all maternal or paternal relatives, respectively. Any comparisons will need to be made between the unknown individual and anyone with a close maternal or paternal relation when attempting to identify human remains with lineage markers (Latham & Miller, 2019). These types of lineage markers are often used when STR analysis is unable to produce results due to DNA degradation

(McNally et al.,1989).

Compared to the nucleus, which has only one copy of a person’s genome, one mitochondrial organelle has multiple identical copies of the mitochondrial genome (Latham &

Miller, 2019). Most human cells contain only one nucleus; therefore, there is only one copy of the nuclear genome per cell. Comparatively, there can be hundreds of mitochondria per cell, each with approximately four to five copies of the mtDNA genome per mitochondrion; therefore, it is estimated that there are about 500 mtDNA molecules per cell (Satoh & Kuroiwa, 1991). As such, there is a higher probability of finding mtDNA over longer periods of time as compared to nuclear DNA. mtDNA’s circular structure, compared to the double helix structure of nuclear

DNA, is more resilient to degradation (Satoh & Kuroiwa, 1991). The value of mtDNA is especially emphasized when the only forensic evidence available are highly degraded samples with little soft tissue or when working with bone samples. 3

Applications for DNA Analysis of Bone

The use of bone for DNA analysis can be of great interest for many different applications. An obvious area is forensic testing. When biological tissue, such as skin or muscle, is unavailable for testing, bone may be the last source of DNA remaining. Cases that involve human remains, found close to the time of death, allow for easier DNA processing compared to those samples that are found weeks, months, or years after the time of death. Natural decomposition starts immediately with the enzymatic breakdown of cells, an autolysis, and eventual bacterial decomposition, termed putrefaction (Schotsmans, Van de

Voorde, De Winne, & Wilson, 2010). Over time, any soft tissue originally adhering to the bone sample, and any DNA found in that tissue, degrades, making autosomal STR analysis difficult.

Although the bone itself may contain significantly less DNA than the tissue originally attached to it, the bone will preserve that DNA for much longer (Wang, Eng, Waye, Dudar, & Saunders,

1998). Large-scale DNA testing, such as missing persons cases or mass fatality scenarios, often have mostly skeletonized remains, requiring specialized bone processing techniques due to the age of the bone being tested. Groups such as the International Commission on Missing Persons

(ICMP) have been working with governments and civil society organizations from around the world, aiding in the identification of individuals using skeletal remains (Parsons, Huel,

Bajunović, & Rizvić, 2019).

Phylogenetic studies also use bone samples to further explain evolutionary developments of species. The Neanderthal, Homo neanderthalensis, is a human subspecies that went extinct about 40,000 years ago. Using a bone sample, researchers were able to develop a complete mitochondrial sequence that was compared to the modern human genome. The data supported previous evidence that modern humans are related to the Neanderthal and helped establish a 4 timeline of when Homo sapiens diverged from H. neanderthalensis. However, how much the

Neanderthal influenced modern human DNA is still under debate (Green et al., 2008).

Bone Structure

There are 213 bones in the adult human body. These bones fall into one of four general categories: long, short, flat, and irregular. Long bones, as the name suggests, are long in length and are the largest bones in the body. The clavicles, which articulate to the sternum, and the femurs in the thighs are examples of long bones. Short bones are the smallest bones in the body and include the carpals in the wrists and the tarsals in the feet. The skull, ribs, and sternum are categorized as protective flat bones. The bones found in the spinal column, such as the vertebrae and coccyx, are examples of irregular bones (Clarke, 2008).

There are three main sections of bone: gross external, gross internal, and microscopic morphology. The gross external portions include the anatomical features of bone that can be seen without any microscopic enhancement. This includes the ends that connect the bone to joints (epiphysis), the shaft of the bone (diaphysis), various prominences that attach to muscles and connective tissues, and openings for blood vessels (foramen) (Byers, 2016).

When a bone is cut open, the gross internal sections of the bone can be seen. The smooth exterior of bone, known as cortical/compact bone, is dense and forms in layers. Compact bone makes up about 80 percent of the human skeletal system and is considered the protective shell to the cancellous bone underneath (Rogers, 2010). Unlike the compact bone, cancellous bone, also called spongy bone, is less dense and porous. Cancellous bone is made up of plates and rods, known as trabeculae, which form open spaces within the bone. This sponge-like bone provides structural support while still giving the bone the flexibility it needs to withstand natural stresses 5

(Byers, 2016). Additionally, long bones, such as the femur, have a medullary/marrow cavity, where bone marrow would be stored.

If the bone is cut into fine cross-sectioned slices and viewed under a microscope, histological features such as the collagen fibers that form the cortex bone can be seen. The vascular canals and osteons, which house blood vessels, can also be seen at this level.

A majority of bone is made up of a matrix that consists of the osteoid (organic portion) and the hydroxyapatite (inorganic portion). The osteoid matrix component is made up of proteins and type I collagen (Collins et al., 2002). Ninety percent of a single bone is made up of fibrils collagen, which is responsible for proper structure maintenance, force transmission, and is what gives bone its tensile strength. The hydroxyapatite [Ca10(PO4)6(OH)2] is made up of calcium phosphate crystals that are the mineral portion of bone, which gives bone its rigid strength and density (Tzaphlidou, 2008).

There are four types of cells that make up the bone matrix: osteoprogenitor, osteoblasts, osteocytes, and osteoclasts. Together, these cells maintain the bone matrix, starting with the osteoprogenitor cells, which are bone stem cells that eventually mature into osteoblasts. The osteoblasts will synthesize collagen, proteins, and enzymes that produce alkaline phosphatase needed for hydroxyapatite (Clarke, 2008). Osteoblasts eventually mature into osteocytes.

Osteocytes can be found in the small spaces in the cortical bone and in the lacunae (the open spaces of bone). These cells contain most of the DNA found in bone (Hochmeister et al., 1991).

Osteocytes are star-shaped, mature bone cells that become embedded within the bone matrix as it forms. Finally, the osteoclasts use enzymes to break down bone for the purposes of creating new bone matrix (Campos et al., 2012). 6

Forensic DNA Testing of Bone

Bone has two main layers: the cortical and cancellous layers. The bone matrix that makes up both layers will house DNA in its osteocyte cells. The physical and chemical barrier that makes up the bone matrix protects the biological material needed for DNA testing, compared to the softer tissue (Loreille, Diegoli, Irwin, Coble, & Parsons, 2007). While the soft tissue surrounding bone, and the DNA within that soft tissue, will decompose over time, the bone itself and the DNA trapped inside can survive long after the death and decomposition of that individual (Kaiser et al., 2008).

Mitochondrial DNA analysis can be the more viable option, compared to nuclear DNA, when attempting to recover DNA from older, degraded samples, such as those from skeletal remains. A study using the remains of the United States service members, missing from past military conflicts concluded that, in concordance with previous studies, extracting mtDNA from weight-bearing bones proved successful for samples ranging in size and state of degradation.

The study did, however, conclude that variables (e.g., exogenous contamination, environmental damage, commingled samples, and handling of the samples by personnel) made the mtDNA testing difficult for some samples (Mundorff, Davoren, & Weitz, 2013).

The environment from which bone samples are recovered from can influence how much

DNA can be recovered. The exact correlation between environmental factors and DNA degradation is still a topic of research given the numerous variables involved. The research also shows that various types of environments will affect DNA degradation in bone samples. For example, high or low temperature and humidity have variable effects on bone DNA preservation. Samples found in colder environments or lower humidity tend to have better DNA 7 preservation compared to locations where there is a constant high temperature or high humidity

(Burger, Hummel, Herrmann, & Henke, 1999). Bone samples found in soil with a high pH have a more rapid biological decomposition. The acidic soil dissolves the bone itself, if given enough time. The pH level also influences the rate of microbial decomposition, making DNA less susceptible to damage in neutral environments (Antinick & Foran, 2019; Latham & Miller,

2019).

Bone found in freshwater or marine environments is attacked by cyanobacteria, responsible for accelerating bone degradation (Campos et al., 2012). If the bone was buried near the surface, it may be vulnerable to the aforementioned variables over time due to seasonal changes, such as freezing, thawing, and exposure to rain (Pietrangeli et al., 2009). In cases where bone samples undergo intense heat due to fire, the bone itself has both physical and chemical alterations, making it very difficult to recover the DNA. The bone matrix becomes severely damaged, leaving the bone fragile and open to contamination (Edson & Christensen,

2013). With special care, it is still possible to obtain DNA from these burned samples, even those that have been cremated (Imaizumi, 2015). Amazingly, DNA can survive a wide range of these exposures for days, months, and even years; however, the amount of DNA that can be extracted will vary significantly.

If investigators have a selection of bone samples from which to choose, selecting the bone, and which section of that bone, that will give the most DNA to produce a DNA profile can be challenging. The type of bone sample being processed (cortical versus cancellous bone) can impact DNA recovery. The quality and quantity of DNA yield and ease of sampling (i.e., accessibility and tools required) should be considered when deciding whether to sample cortical or cancellous bone. Current practice for choosing bone samples is based primarily on the 8 experience of practitioners in the field. In Europe, the Interpol Disaster Victim Identification

Guide, which over 80 nations follow, suggests the collection of compact/cortical bone from long bones (Interpol, 2015). Similarly, the National Association of Medical Examiners has an entire protocol for mass disasters, advocating for the selection of long bones and teeth when retrieving samples from decomposed remains. However, guidelines cannot account for every scenario, such as still intact remains compared to fragmented and burned samples. Some of the current guidelines, such as those from the National Institute of Justice (NIJ) and the International Society of Forensic Genetics (ISFG), do not address which skeletal samples are likely to result in higher

DNA recovery but rather provide general sampling recommendations (Mundorff, Bartelink, &

Mar-Cash, 2009).

Current research suggests that the traditional sample selection of cortical bone may not be as clear as once believed. A previous study attempting to examine the variations of osteocyte lacunar properties between cortical and cancellous bone found that cancellous bones, such as the tarsals and patella, have actually yielded higher quantity and quality DNA than the weight- bearing, long cortical bones traditionally chosen for DNA testing (Andronowski, Mundorff,

Pratt, Davoren, & Cooper, 2017). This same study viewed and created three-dimensional (3D) scans of cancellous and cortical bone by utilizing a micro-Computed Tomography lens, which uses X-rays to collect a magnified image in an attempt to observe the lacunar properties in the bone sample. The 3D scans consistently showed remnants of soft tissue within the spaces of skeletal elements of high cancellous content. This was even true for samples that had no soft tissue at the surface of the bone. It was hypothesized that bone marrow cells and bone lining cells filling in the porous structure that characterizes cancellous bone contributed to the higher

DNA yields (Andronowski et al., 2017). This may be why other studies found that the smaller, 9 primarily cancellous bones, such as the patella and phalanges, exhibited very high success rates for DNA analysis (Hines et al., 2014).

Data from studies involving large-scale identification projects have corroborated the viability of processing cancellous bone by confirming that such samples can result in similar or better DNA yields than cortical bone. One study, which used the World Trade Center Remains

Database (n=3,631) to evaluate the DNA preservation in bone samples based upon the skeletal type, documented that lower limb bones, such as the femur or tibia (which tend to be denser and thicker cortical bone samples), were more likely to generate DNA profiles, with a success rate of

77% and 71% respectively. However, it was also noted that cancellous bones, especially the lower limb bone samples encased in soft tissue like the phalanges of the feet, the metatarsals, and the patella, resulted in successful DNA profiles (86%, 80.8%, and 80% respectively) that were comparable or better than that of the cortical bone samples (Mundorff et al., 2009).

A similar study attempted to create a ranking order for bone sampling by comparing the success of DNA yield rates between skeletal elements over increasing post-mortem intervals.

After selecting representative sample types (n=55) from 3 recently skeletonized donors in the study’s first phase, it was concluded that the smaller cancellous bones, such as the phalanx in the hand, had the highest DNA yields (~448 ng DNA/g) compared to some of the larger bones, such as the tibia (~47 ng DNA/g). In the study’s second phase, data collected from the first phase was used to determine which samples to test from skeletons with post-mortem intervals ranging from

3 years to over 20. A total of 10 bone types from 3 individuals were tested. The results were approximately the same as those of the first phase, even from the oldest samples, suggesting that cancellous bone samples may be a better option for DNA testing (Mundorff et al., 2013). 10

Both Mundorff studies also noted that along with high DNA yields, samples made primarily of cancellous bone, such as the phalanges, are generally more accessible, require less work to sample, and often can remain completely intact when initial testing begins as compared to the larger cortical bones. The use of smaller cancellous bone samples allows for the use of simpler tools (i.e., tools that do not need to be electrically powered), and lower the potential for contamination (Mundorff et al., 2009). Reducing contamination is important, which is why using intensive mechanical tools to cut into the large weight-bearing bones may be less preferable than sampling the smaller cancellous bones that can be removed more easily, in some cases with just a single-use blade—which may be the only tool available depending upon the mortuary operations of the location. Ultimately, cancellous bone samples can be a viable alternative to cortical bone samples for DNA testing.

Methods for Processing Bone

Prior to DNA extraction, bone samples must go through a maceration processing stage in which any remaining tissue is removed from the outer layer before tissue lysis. The early maceration step of bone DNA processing can vary but is crucial and can have major impacts on the quality and quantity of DNA that is recovered (Higgins, Rohrlach, Kaidonis, Townsend, &

Austin, 2015). The maceration stage is vitally important because samples can be inundated with foreign biological material both from their recovery site, such as soil and bacteria, and through physical contact in the field or while in the lab (Ogden, Dawnay, & McEwing, 2009). The presence of PCR inhibitors and bacterial contamination from exogenous sources must also be considered, as those will interfere with downstream forensic testing (Sampietro et al., 2006). 11

Older bone samples, or samples found in extreme conditions, such as samples that were submerged in water, buried in soil, or layered with putrefying remains not associated with the sample, are at risk of having more than just the surface being contaminated (Zehner, 2007).

Contaminants can even enter the bone through the porous layers and affect any DNA testing performed (Gilbert et al., 2005). All these factors can make it more difficult to obtain amplifiable DNA, which underscores the importance of the maceration step in removing any exogenous DNA that can overwhelm the endogenous DNA that is targeted for sequencing (von

Wurmb-Schwark, Heinrich, Freudenberg, Gebühr, & Schwark, 2007).

A common type of bone preparation involves the manual removal of the outer surface layer of the bone. The outer layer can have soft tissue remaining from when it was collected.

This material can be sanded down manually using sandpaper, however this is, by far, the most laborious and time-consuming method (Anslinger, Weichhold, Keil, Bayer, & Eisenmenger,

2001; Bender, Schneider, & Rittner, 2000; Iwamura, Oliveira, Soares-Vieira, Nascimento, &

Muñoz, 2005). Another type of bone preparation includes the use of mechanical tools, such as a rotary tool (Courts & Madea, 2011; Davoren et al., 2007; Edson, Ross, Coble, Parson, & Barritt,

2004). This mechanical method may be too destructive for bones that are small and fragile, mainly cancellous, or highly degraded. A manual sanding method would need to be used to avoid destroying the sample in such instances (Edson et al., 2004).

These well-established preparation methods do, however, have some processing disadvantages. One such disadvantage is the abrasiveness of the method being used. Softer, more fragile samples of bone may not withstand certain mechanical tools. These methods also create bone dust, which makes simultaneous sample testing very difficult and requires increased care to limit the possibility of contamination. Bone dust can also be a health hazard, potentially 12 exposing anyone to blood-borne pathogens. The bone dust itself can contaminate the laboratory and any samples that are not carefully stored (Budowle, Bieber, & Eisenberg, 2005; Li &

Klempner, 2013). This usually means limiting the number of samples that can be tested at the same time (Kitayma et al., 2010). Safety measures must be put in place for any of the physical methods as they will create bone dust that will become airborne.

Other methods to remove contaminants include extracting the sample from only the interior of the bone by removing a section with a saw and scraping the outer and inner surfaces, leaving the interior of the bone to be sent on for further testing (Palmirotta et al., 1997). Like other manual methods, this involves the use of destructive tools that can damage the overall bone sample.

There are even more extreme methods to remove unwanted material from a bone sample, such as the use of insects. The method uses a species of insect, such as the carpet beetle, to literally eat away tissue found on the bone. Although quite effective, especially for small or delicate bones, this method would require thousands of insects, an area to contain them, and constant maintenance because these insects would begin to eat the bone if deprived of material to consume (Mairs, Swift, & Rutty, 2004).

The use of a liquid-based maceration technique helps circumvent the disadvantages of traditional manual methods. The risk of damaging the bones with mechanical tools would be completely avoided, along with the potential for contamination due to bone dust. Previous studies have demonstrated a range of liquid maceration techniques, including bleach. One maceration method previously studied is treatment with ultraviolet light for at least 30 minutes following the removal of bone surface materials with a liquid washing in bleach and water in 13 order to aid with decontamination (Kalmár, Bachrati, Marcsik, & Raskó, 2000). This method serves more as an additional measure than a stand-alone method for contaminant removal.

Macerating crushed bone in a bleach solution has also been tested as a means to further reduce the amount of exogenous DNA on the bone surface; however, additional cleaning methods are then needed prior to DNA extraction to make the bone bleaching step more effective (Carlyle,

Parr, Hayes, & O’Rourke, 2000).

One of the least chemically invasive maceration methods is to simply wash the bone briefly in distilled water and ethanol before a sample is taken (Eagle, Man, Rooney, Hogg, &

Kearney, 2015). With so many other chemical options, the use of water and ethanol alone would be a poor choice and would still require an additional step, such as sanding, to ensure a bone has been well macerated.

One of the oldest and most basic maceration techniques is the use of warm water. This method relies on bacterial decomposition of the tissue attached to the bone. This method is simple and requires little in materials, other than adequate ventilation due to the smell; however, the process could take between two days and eight weeks, depending on the temperature of the water, amount of bacteria, and size of the bone (Ator, Andrews, & Maxwell, 1993).

If time is an important factor, as it often is with forensic cases, other liquid maceration methods have also been developed as more efficient alternatives, while also reducing the associated smell. These maceration methods make use of detergents and enzymes found in cleaning agents to remove tissue in only a few hours compared to days or weeks of older methods (Offele, Harbeck, Dobberstein, Wurmb-Schwark, & Ritz-Timme, 2007). The use of detergents often requires using commercial grade options, which can contain additives, bleaching 14 agents, corrosion inhibitors, and enzymes. Because these commercial options often do not include a full breakdown of their proprietary ingredients, the unknown mixture can cause damage due to decalcification, softening, and even cause transparency of the bone (Fenton,

Birkby, & Cornelison 2003). If the ultimate goal is to obtain a bone sample that could be examined for knife or toolmark analysis, this type of damage can be extremely problematic

(King & Birch, 2015). If the purpose was to isolate DNA from these samples, these detergents’ unknown ingredients can damage the DNA within the bone sample (Uhre et al., 2015).

As DNA has become a vital tool for forensics, anthropology, and species identification, being able to obtain DNA from bone samples has become increasingly important. As a result, maceration methods have been developed to have better control of the chemicals utilized so as to not damage the DNA being extracted. Studies have investigated how maceration methods using detergents, proteases, lipases, chemicals, or a combination thereof have affected DNA recovery.

Steadman et al. (2006) tested several maceration techniques taken from various published and unpublished literature, including bleach, hydrogen peroxide, and ethylenediaminetetraacetic acid

(EDTA) combined with the proteinase papain. The study wanted to compare the different methods’ ability to macerate bone and their effect on the recovery of nuclear and mtDNA from pig bone. The results of the study concluded that as a means of maceration, the enzyme and detergents produced clean bone samples in a much faster time frame (10 days) when combined with high temperatures (45 oC) compared to the water bath control (23.9 days; 22 oC) alone.

However, boiling water (100oC) was able to macerate faster (14 hr) than both of those options.

As for DNA recovery, the Steadman et al. (2006) study concluded that the best methods used a meat tenderizer (Adolph’s®) combined with a detergent (Palmolive®) and a detergent (Biz®) combined with sodium carbonate at a high temperature (90 oC). Each of these methods were 15 able to recover more mtDNA (1.17 µg/µL, 1.60 µg/µL) compared to the bleach (0.10 µg/µL), hydrogen peroxide (0.0 µg/µL), and EDTA/papain (0.06 µg/µL) methods. Of the tested maceration methods, nuclear DNA was successfully amplified only after maceration in the meat tenderizer coupled with detergent and the detergent combined with sodium carbonate; mtDNA was successfully amplified after maceration in all methods (Steadman, DiAntonio, Wilson,

Sheridan, & Tammariello, 2006).

Similar results were obtained in a follow up study by Lee et al. (2010) that focused on nuclear DNA testing of human bone samples using the same techniques compared by the

Steadman et al. (2006) study. The results corroborated those of Steadman et al. (2006), including that bleach makes a poor chemical choice for maceration purposes. The study also concluded, however, that the EDTA/papain combination was successful when left at lower temperatures (45oC) over 4 days, which contradicts the results of the previous study for pig bones. Lee et al. (2010) also theorized that at higher temperatures (90oC), the EDTA/papain method may be a faster and possibly more successful method than the Adolph’s® and

Palmolive® method but that it was not feasible because the papain enzymes would denature at that temperature.

Another enzymatic maceration method uses trypsin, a proteolytic enzyme, which has been observed to remove soft tissue attached to bone for both pig and human samples. The study tested varying temperatures (37, 45, 55, 65 oC), trypsin concentrations (0, 0.3, 1, 3, 10, 30 µg/µl), and incubation times (0, 15, 30, 45, 60, 120 min) to find the optimal settings for maceration by observing the proteolytic activity. The study concluded that the most proteolytic activity was observed at higher concentrations of trypsin (30 µg/µL) at 55 oC for 2 hours. Human and pig bone samples were then tested at these optimal settings to determine if DNA could be amplified. 16

For both species of sample, DNA was successfully amplified. The results demonstrated that trypsin could be used as a maceration technique for samples prior to DNA isolation (Li,

Chapman, Thompson, & Schwartz, 2009). A study from the same author used the same maceration method to test if non-human bones could successfully be sequenced following processing with the trypsin method. The study was able to successfully sequence a segment of mtDNA (cytb) for both treated and non-treated samples, demonstrating that the maceration method had no adverse effect on downstream sequencing (Li & Liriano, 2011).

Another study specifically testing for mtDNA found that a combination of a protease

(Savinase®) and a lipase (Lipex® 100L) with high temperature (55oC) incubations was able to sufficiently macerate bone of a Stone Marten (Martes foina), followed by successful sequencing of mtDNA for species identification. Interestingly, the extraction and DNA testing was not performed until two years after the initial maceration, suggesting that the protease and lipase did not penetrate into the bone during the process and degrade DNA within the bone (Eriksen,

Simonsen, & Rasmussen, 2013).

Focus and Goals

One of the primary goals of this study was to address the issues associated with manual maceration methods. Prior to DNA isolation, biological materials present on the bone samples need to be removed, such as PCR inhibitors and bacterial contamination, as they will interfere with downstream DNA analysis (Calacal & De Ungria, 2005). This is especially true for commingled remains (Zehner, 2007). Manual sanding by hand or with mechanical tools is laborious and time-consuming and will create bone dust that can potentially lead to cross- contamination between samples and require more equipment to contain the dust (Kitayama et al., 17

2010). The potential for contamination between samples when using manual maceration methods also makes testing multiple samples at once very difficult. This study proposes a method that will eliminate the need for an abrasive tissue removal step by replacing manual maceration with a liquid-based maceration method that uses a combination of a proteinase

(proteinase K), an acid (EDTA), and a cleaning agent (1% lauryl-sarcosinate). The effectiveness of this unique solution as a maceration method is tested using short incubation times at high temperatures, followed by a tissue disruption step that has been seen in previous studies to aid in

DNA recovery from macerated samples (Franchi, Pilli, Barni, Potenza, & Berti, 2011).

It is of particular note that this method allows for the maceration and tissue lysis steps to be performed in a single tube, which enables the testing of multiple samples at one time. This novel method allows for the possibility of high-throughput testing and automation, an option not possible with standard manual maceration methods. Emergency situations that require mass testing, such as a mass disaster, would greatly benefit from a maceration method that could be automated.

Additionally, this study focuses on the use of cancellous bone, the less dense and porous bone that contains open spaces found under the thicker cortical bone. The choice between sampling cortical or cancellous bone can potentially impact DNA recovery; however, because every scenario is different, it is difficult to create all-encompassing guidelines for investigators to follow. Variables such as the age and condition (intact, fragmented, burned, etc.) of bones can affect sample selection. Many of the available guidelines from groups such as the ISFG and the

NIJ only give recommendations that generally support the use of cortical bones for sample collection; however, studies discussed previously have observed that cancellous bone can be as viable as cortical bone for DNA isolation. It was also noted that samples made primarily of 18 cancellous bones, such as the phalanges and ribs, were easier to sample, in some cases as simple as using a single blade. The Andronowski et al. (2017) study went as far to theorize that the marrow cells found within the cancellous bone layer could contribute to higher DNA yields.

The present study utilizes swine (Sus Scrofa) bones, a useful model for simulating human bones, to test for both quantity and quality of mitochondrial DNA recovered from samples that undergo the proposed liquid-based maceration method, focusing on the use of cancellous bone as a viable alternative to cortical bone samples for DNA testing.

Lastly, this study assesses the effect of the liquid-based maceration method on subsequent species identification of samples. Samples from different species are processed using the maceration method and then undergo mtDNA testing. The resulting data is compared to a known BLAST database to determine if application of this method aided in the generation of mtDNA of sufficient quality and quantity for species identification. The scope of this study will be testing a single-tube liquid maceration method, a viable alternative to manual maceration, while also demonstrating the viability of cancellous bones as a source of mtDNA with the intention of species identification.

19

Materials and Methods

Sample Procurement

Due to their usefulness as a model system for simulating human bone, swine (Sus scrofa) rib bones were used in this study (Figure 1). Soft tissue on the bone surface was removed with a razor. The bones were dissected using a rotary tool (Dremel, Racine, WI). Bovine, swine, chicken, duck, sheep, and fish bones were obtained from food stores for animal species testing.

Aged human rib bones (approximately 50 years post-mortem), previously collected (Li &

Klempner, 2013), were used for human sample testing. Standard precautions were followed to reduce any possible DNA contamination. All sampling was carried out in a sterilized laminar flow cabinet. 20

Figure 1. A typical swine rib bone fragment specimen utilized in this study.

21

Bone Sample Maceration

The maceration treatment of bone fragments (approximately 0.1 g) was carried out in 1.6 mL of a proteinase K solution (0.5 M EDTA, 1% lauryl sarcosinate, and 2 mg of proteinase K;

Fisher Scientific, Waltham, MA) for a 1 to 2 hr incubation at 56°C with gentle agitation. The bone fragments were further processed by inversion for 30 sec in distilled water, 0.5% sodium hypochloride and 96% ethanol, as described in Davoren et al. (2007). The bone fragments were then air dried, in tubes, inside of a sterilized laminar flow cabinet. The macerated bone samples were examined using light microscopy, confocal imaging, and digital photography.

Bone Tissue Disruption

The tissue disruption of swine bones was performed by grinding. The tissue disruption of bone samples for species identification was carried out based on the following procedure (J. Fox, personal communication). Bone fragments, approximately 0.1 g, were placed into a 2-mL flat- bottomed microcentrifuge tube that contained a sterilized 5-mm stainless steel bead. Sample tubes were placed into an Adapter Set 2 x 24 and loaded into the TissueLyser II (QIAGEN,

Valencia, CA). Initial disruption for all samples was set for 20 min at 30 Hz. Tubes were examined and then rotated. Additional 10-min disruption cycles were applied as needed until bone samples were a fine powder. Total disruption time for cancellous bone fragments was 20 min and 30 min for bone samples with both cancellous and cortical components.

DNA Extraction and Quantification

22

The demineralization of the bone powder for all samples was performed as described in

Loreille et al. (2007). Each sample (approximately 0.1 g) of the pulverized bone was placed in

1.6 mL of extraction buffer (0.5 M EDTA, 1% lauryl sarcosinate) and 200 µL of 20 mg/mL of proteinase K. Samples were incubated overnight at 56oC with gentle agitation. The samples were then concentrated by reducing the total volume to approximately 200 µL using Amicon Ultra-15

(30 kDA) columns (Millipore, Billerica, MA). The membranes used in this column system are characterized by their molecular weight cutoff, i.e., their ability to retain molecules. The

Amicon Ultra-15 column allows for a greater number of short DNA elements to be collected because of the filter column’s small molecular weight cutoff, which increases the potential for successful amplification (Gabriel, Huffine, Ryan, Holland, & Parsons, 2001).

The DNA was extracted using QIAamp DNA Micro Kit (QIAGEN, Valencia, CA). For each sample, 200 µL of buffer AL added followed by a 15-sec vortexing, creating a homogenous solution. 200 µL of ethanol (96%) was added followed by another 15-sec vortexing. Tubes were incubated for 5 min at room temperature then centrifuged to remove drops from the lid.

Each sample had their entire lysate carefully transferred to their own QIAamp MiniElute column

(inside a 2-mL collection tube) and centrifuged at 8000 rpm (6000 x g) for 1-min. The column was removed and placed into clean 2-mL collection tube and the centrifuge step was repeated until the lysate had completely passed through the column membrane, discarding the flow- through each time. Once the column was empty, 500 µL of Buffer AW1 was added and the column was centrifuged at 8000 rpm (6000 x g) for 1-min. The column is then placed into a clean 2-mL collection tube, discarding the flow-through, and 500 µL of Buffer AW2 is added and centrifuged again at 8000 rpm (6000 x g) for 1-min. The column is placed into another clean

2-mL tube, discarding the flow-through, then centrifuged at 14,000 rpm (20,000 x g) for 3-min to 23 completely dry the membrane. The column is then placed into a 1.5 mL microcentrifuge tube and 50 µL of Buffer AE was added. The column is incubated at room temperature for 1-min then centrifuged at 14,000 rpm (20,000 x g) for 1-min. The final elution volume was 50 µL. To monitor potential contamination, extraction negatives were employed. For non-human DNA samples, quantitation was performed using the NanoDrop 2000c spectrophotometer

(ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s protocols. Human

DNA samples were quantified using the Quantifiler Human DNA Quantitation kit

(ThermoFisher Scientific, Waltham, MA) on an ABI 7300 Real Time PCR system. For each reaction, 8 L of Quantifiler Trio Primer Mix and 10 L of Quantifiler THP PCR Reaction Mix were used. Additionally, 2 L of the DNA sample to be quantified was added. Total reaction volume was 20 L. The reactions were run with a dilution series of the DNA standard provided by the kit at 50, 5, 0.5, 0.05 and 0.005 ng/l in duplicate. The quantification was performed using MicroAmp Optical 96‐Well Reaction Plates (Thermo Fisher Scientific, Waltham, MA).

Thermal cycling was performed using the following thermal cycle: 95oC for 2-min followed by

40 cycles of 95oC for 9-sec; 60oC for 30-sec. HID Real‐ Time PCR Software v1.2 (Thermo

Fisher Scientific, Waltham, MA) was used for the collection and the analysis of the quantitation data.

Species Identification

For swine species identification, sequencing of specific areas on the mitochondrial cytochrome b (cytb) locus was performed. PCR reactions were performed in volumes of 25 µL.

Each set contained GeneAmp PCR Gold Buffer (Applied Biosystems, Foster City, CA), 1.5 mM 24

MgCl2, 200 µM of each dNTP, 1 mM bovine serum albumin (Sigma-Aldrich, St. Louis, MO), 2 units of AmpliTaq Gold DNA Polymerase (Applied Biosystems), and 0.4 µM of each forward

(5’-TCA CAC GAT TCT TCG CCT TCC ACT-3’) and reverse (5’-TGA TGA ACG GGT GTT

CTA CGG GTT-3’) primer that amplifies the cytb gene in Sus scrofa (Steadman et al., 2006).

The swine mitochondrial cytb gene amplicon was expected to be 521 bp long (nucleotide position: 524-1022; GenBank accession number: AY237533). PCR involved an 11-min activation step at 95oC, followed by 34 cycles consisting of a 30-sec denaturation step at 94oC,

30-sec primer annealing step at 50oC, and a 30-sec extension step at 72oC. A half nanogram sample of known swine mitochondrial DNA was used as a positive control and a PCR negative control was included for each amplification process.

PCR amplification of the COI gene was carried out using the following primers: vertebrate forward primer (5'-TCT CAA CCA ACC ACA AAG ACA TTG G-3'), vertebrate reverse primer (5'-TAG ACT TCT GGG TGG CCR AAR AAY CA-3'), fish forward primer (5'-

CAA CCA ACC ACA AAG ACA TTG GCA C-3'), and fish reverse primer (5'-ACT TCA GGG

TGA CCG AAG AAT CAG AA-3'). PCR amplification was carried out under the following cycling parameters: an initial step (1 min at 94oC) followed by 35 cycles (denaturing, 15 sec at

94oC; annealing, 15 sec at 54oC; and extending, 30 sec at 72oC).

Amplified samples were examined by electrophoresis using a 1% agarose gel utilizing a

100 bp ladder to estimate amplification yields. Amplified samples were sent to GENEWIZ in

South Plainfield, NJ for post-PCR clean up and Sanger sequencing. The sequence files were viewed, trimmed, and edited using the FinchTV 1.5 chromatogram viewer. One value the program displays is the Quality Value (QV), which is inversely proportional to the probability that a called base has been misidentified. Therefore, the higher the QV, the higher the 25 confidence that the base call is correct. A QV value of 20 indicates base calls that have a 1% chance of being identified incorrectly. The 5’ and 3’ ends were trimmed until the first 10 bases had less than 1 base with a QV value less than 25. BioEdit 7.2 was used to create the sequence alignment and contig assembly. The BLAST database was used to search the contig sequences to identify the samples’ species.

Human mtDNA Sequencing

The sequencing of the hypervariable region 1 (HVI) was performed. The target amount of DNA to be amplified was 0.1 ng DNA per 20 µL. Samples were amplified using the following primers: HV1 forward primer (position 15975; sequence 5’-

CTCCACCATTAGCACCCAA-3’), and HVI reverse primer (position 16418; sequence 5’-

ATTTCACGGAGGATGGTG-3’). PCR amplification was carried out under the following cycling parameters: an initial step (14 min at 94oC) followed by 34 cycles (denaturing, 15 sec at

92oC; annealing, 30 sec at 59oC; and extending, 30 sec at 72oC; adenylation, 10 min at 72oC).

AmpliTaq Gold 360 kit (Thermo Fisher Scientific, Waltham, MA). Amplified samples were examined by electrophoresis using a 1% agarose gel utilizing a 100 bp ladder to estimate amplification yields. Amplified samples were treated with ExoSAP-IT PCR Product Clean-Up

(Thermo Fisher Scientific, Waltham, MA) reagent to deactivate any unincorporated primers and nucleotides still present in the samples.

For DNA sequencing, the Big Dye Terminator Big Dye® and Terminator v3.1 Cycle

Sequencing Kit (ThermoFisher Scientific, Waltham, MA), was used. Samples were sequenced in the forward direction (position 15978; sequence 5’-CACCATTAGCACCCAAAGCT-3’) and 26 reverse direction (position 16410; sequence 5’-GAGGATGGTGGTCAAGGGAC-3’). Cycling conditions were as follows: (soak, 1 min at 92oC), 25 cycles (denaturing, 15 sec at 96oC; annealing, 1 sec at 50oC; and extending, 1 min at 60oC). Following the manufacturer’s protocol, sodium lauryl sulfate (SDS) and DyeEx 2.0 were used for post-cycle cleaning followed by vacuum-drying with the SpeedVac system (ThermoFisher Scientific, Waltham, MA). Prepared sequencing standards, controls, and samples were run on the 3500 Genetic Analyzer (Thermo

Fisher Scientific, Waltham, MA). Samples were processed using the ABI Sequencing Analysis software and then imported into the Gene Codes Sequencher (Gene Code Corp., Ann Arbor, MI) alignment software to create a consensus sequence that was compared to the rCRS sequences.

27

Results and Discussion

Macroscopic and Microscopic Effects of Specimen Maceration

The maceration process began with an incubation in a buffer, containing proteinase K,

0.5 M EDTA and 1% lauryl-sarcosinate. Proteinase K, a serine protease, typically used for digestion of native proteins, was employed during protein digestion steps. The amino acid sites that proteinase K targets are only minor components of bone’s collagen, which is why proteinase

K is often used in addition to detergents for digestion purposes (Barrett, 2015). EDTA finds uses in both industrial and medical fields. EDTA is a chelating agent, used in detergents, and is an effective method for bone cleaning (Mairs et al., 2004). Lauryl-sarcosinate, a surfactant, lowers surface tension between solids and liquids, aiding in the removal of solids in liquids. Surfactants also act as detergents and inactivating agents, inactivating or killing viral, bacterial, or fungal contamination (Reichl, 1997).

The outer layer of the enzyme-treated bone fragments was visually examined after each incubation period to observe any morphological changes to the bone surface. Using a digital single-lens reflex (DSLR) camera, cross-section images of the bone surfaces were taken. Figure

2 depicts the appearance of the bone samples immediately after the samples were removed from their allotted incubations. Compared to the untreated bone, the 1-, 1.5-, and 2-hr incubations showed a clear degradation of bone surface. The removal was more prevalent in the cancellous bone as compared to the compact bone. As the bone incubation time increased, the spongy structure trabeculae within the cancellous bone appeared to get larger, indicating that some of the cancellous bone had been dissolved. After one hour, the bone began to form depressions in both 28 the cortical and cancellous layers. After two hours, these depressions became even larger, with some becoming small holes.

29

Figure 2. Digital single-lens reflex camera observation of bone surface (swine). Bone degradation was compared: without treatment (A), and after 1-hr (B), 1.5-hr (C), and 2-hr (D) treatment. Field width: 3.5 mm.

30

Macroscopic observations were consistent with observations made with light microscopy.

Compared to the untreated compact bone layers, which appeared as a solid layer, the treated compact bone layers began to develop depression pits and small openings as the incubation times increased. These openings began to resemble the openings seen in the trabeculae matrix in the cancellous bone layer beneath the compact bone. The compact bone structure after the 2-hr incubation appeared porous and dissolved in some areas (Figure 3).

Confocal imaging was used to further assess the maceration and degradation of the bone surface following each incubation period. Confocal laser scanning microscopy (CLSM) utilizes a laser instead of a standard light source and can move (or scan) across the surface of an object to create entire 3D images of the object being examined (Tata & Raj, 1998; Vorburger, Song & Petraco,

2015). The 3D CLSM imaging in Figure 4 demonstrates the changes in bone surface between the untreated and treated samples. Compared to the smooth, untreated surface of the control sample, the treated samples became progressively more rough and uneven the longer they were incubated. These changes are consistent with the DSLR and light microscopy images that showed a degradation of the cortical and cancellous layers of the incubated samples. Since the bone itself had displayed varying levels of degradation following incubation, this suggests that any surface material, including any remaining soft tissue, would also have been removed. 31

Figure 3. Light microscopy observation of bone surface (swine). Bone degradation (arrow) was compared: without treatment (A), and after 1-hr (B), 1.5-hr (C), and 2- hr (D) treatment. Field width: 3.5 mm.

32

Figure 4. Confocal images of bone samples surfaces. Bone degradation was compared: without treatment (A), and after 1-hr (B), 1.5-hr (C), and 2-hr (D) treatment.

33

Effects of Specimen Maceration on DNA Yields

To observe the effect of the enzyme-based maceration method on the quantity of the isolated DNA, the DNA extracted from untreated swine bone and swine bone treated with the enzyme method was compared. Four trials of experiments (n=4) were carried out. The DNA yields of the samples were quantified to assess the impact of the maceration method. The average DNA yield across all treated samples was slightly lower than that of the untreated samples. A single-factor ANOVA was conducted to compare the average yields of the incubation periods to determine if there were any statistically significant differences in DNA recovery. Based on the ANOVA analysis, there was no significant difference in the yields of these samples across treatments (Figure 5), suggesting that the enzyme-based maceration method does not significantly affect DNA recovery from bone samples.

34

Figure 5. DNA Yields. Four trials of experiments (swine bones) were carried out. The average DNA yields of treated samples were slightly lower than that of untreated samples. A single-factor ANOVA was conducted to compare the yields of the samples. There was no significant difference in the yields of these samples (p value = 0.5219).

Effects of Specimen Maceration on DNA Quality

Using mitochondrial DNA analysis, direct sequencing was used to evaluate the quality of the DNA isolated from the macerated bone samples. The amplified fragment of the mitochondrial cytb locus was performed and the resulting electropherograms were compared side-by-side. The ISFG defines the hallmark of a mtDNA mixture to be overlapping peaks in the electropherogram, requiring letter designations based on which bases are overlapping (Parson et al., 2014). Comparison of the amplified cytb locus and the sequenced bone samples showed defined peaks, with no overlapping seen, which indicates a single source sample free of contamination or adverse effects from the enzyme treatment on the bone samples (Figure 6).

The DNA sequence alignment was viewed for the swine mitochondrial cytb locus and no 35 contradictions were seen between the control samples and any of the experimental samples

(Figure 7).

36

Figure 6. Direct sequencing of amplified fragment at swine mitochondrial cytb locus. The electropherograms of A) the untreated control sample, B) 1 hr, C) 1.5 hr, and D) 2 hr treated sample. 37

Figure 7. The sequence alignment of DNA contig sequences at swine mitochondrial cytb locus. The contig sequences of untreated control sample, 1-hr, 1.5-hr, and 2-hr treated sample are shown. Each contig sequence was obtained by grouping forward and reverse sequences together to produce a consensus. 38

The resulting DNA sequences were searched using BLAST to compare to the reference sequence database. The results of the BLAST search are summarized in Table 1. Scores were provided for each search, which correspond to the number used to assess the biological relevance of the findings, i.e., the overall quality of the alignment. The higher the score value, the higher the similarity between the sequence being searched and the reference sequence in the database

(Tatusova et al., 2013). All treated samples resulted in a score greater than 900. The score value is also inversely proportional to the Expect value (E-value) provided. The E-value is the number of distinct matching alignments that would be expected to occur in a database search by chance.

The lower the E-value, the more significant the score value is. The BLAST tool will assign any

E-value less than 1.0x10-179 as a 0. All tested samples resulted in an E-value of 0, meaning the score of 900 can be considered significant. Lastly, the percent identity for each tested sample was 100%. This number describes how similar the sequenced DNA sample is to the target sample, in this case: Sus scrofa. A percent identity of 100%, an E-value of zero, and a score of greater than 900 all confirm that the samples did originate from Sus scrofa. The use of the enzyme-based maceration method resulted in quality DNA, which allowed for the accurate identification of the Sus scrofa samples.

39

Table 1. Summary of the sequence identification results at cytb locus using GenBank BLAST. The sequence identification results of untreated control samples, and 1-hr, 1.5-hr and 2-hr treated samples are shown.

Sample Species Alignment Length Score E-Value Percent. Identity (%) Identified (bp)

Control Sus scrofa 502 928 0.0 100

1 hr Sus scrofa 494 913 0.0 100

1.5 hr Sus scrofa 503 928 0.0 100

2 hr Sus scrofa 494 913 0.0 100

In-Tube Bone Tissue Disruption

Tissue disruption of bone samples is necessary to improve DNA yield (Rothe & Nagy,

2016). For instance, the application of liquid nitrogen to allow for manual disruption in bones has been reported (Carter, Kilroy, Gimble, & Floyd, 2012; Hasap et al., 2020). The tissue disruption method used in this study was that of Fox (see Materials and Methods). The method can process bone fragments of cancellous bones in 2-mL microcentrifuge tubes (J. Fox, personal communication). Therefore, the same microcentrifuge tube containing a macerated sample can then be used for tissue disruption--without changing tubes. Figure 8a shows a typical bone fragment containing a cancellous portion. The cancellous portion of the bone sample was then dissected and disrupted. Figure 8b shows the bone sample after tissue disruption. Cancellous bone fragments were observed to be broken down to powder.

40

Figure 8. Tissue disruption of cancellous bone fragments. A) A typical swine bone fragment, cancellous portion is shown (arrow). B) Bone powder observed after tissue disruption.

The concept of a single-tube bone processing method was developed in this study.

Figure 9 shows the flow chart for isolation of DNA from bone samples. This study suggested that Steps 1 to 3 can be done in a single microcentrifuge tube without needing to change tubes.

Following the proteinase K, EDTA, and lauryl-sarcosinate incubation, samples go through additional cleaning using water, 0.5% sodium hypochloride, and 96% ethanol to help clean the surface further. This additional step also has the potential benefit of allowing the cleaning solutions to work into the bone itself through the naturally occurring bone canals. The samples are then processed for tissue disruption. The use of the Qiagen TissueLyser system for bone disruption allows for multiple samples to be processed at once, thereby increasing the overall 41 throughput of the method. Samples are then left to dissolve in buffer, containing 0.5 M EDTA,

1% lauryl-sarcosinate, and proteinase K. Once processed, DNA extraction, quantitation, and analysis can be performed using standard methods (see Materials and Methods). The proposed method is intended to allow for sample maceration and tissue disruption using a single tube in order to reduce the need for any transfer steps. This method is designed to reduce contamination risks and sample mix-ups, and to potentially allow for high-throughput testing.

42

Figure 9. Flowchart of procedure for isolating DNA from bones.

43

Animal Species Identification

From a taxonomic point of view, species identification is one of the most important tasks in biology. Research studies require that an organism is first correctly identified. Cataloging different species using DNA helps build data beyond simply trying to identify a species through observation alone. DNA can also be used to compare genetic diversities to resolve taxonomic uncertainties (Arif et al., 2011).

Wildlife forensics has many similarities to the type of testing performed for human DNA analysis. The application of DNA profiling to legal cases involving wildlife crimes is an ever- increasing local and international field. Investigations involving illegal hunting and illegal trade of protected and endangered wildlife are often contingent on DNA evidence (Moore & Frazier,

2019). Wildlife forensics often needs to determine if a crime has been committed and, to do so, will need to identify a particular species. Samples that have no recognizable morphological features are completely dependent upon DNA for identification (Jun et al., 2011). Wildlife identification can even be an effective tool for tracking adulterations in food products (Staats et al., 2016).

The locations most commonly used for species identification are the mitochondrial cytb, cytochrome c oxidase I (COI), and displacement loop loci (Linacre et al., 2011; Herbert

Ratnasingham, & De Ward, 2003). The cytb locus has been favored because of its use in taxonomy and species identification, specifically for the identification of vertebrates, ranging from sharks to rhinos (Chapman et al., 2003; Hsieh et al., 2001; Linacre et al., 2011; Wang et al.,

2003). The “barcode” methodology of cataloging the planet’s species involves sequencing about

650 base pairs of the mitochondrial COI gene, systematize these sequences, and comparing 44 unknown species to known databases. While used for invertebrates somewhat heavily, there are studies that show that COI can be used similarly to cytb for discrimination between closely allied species. Diversity in the amino acid sequences of the COI locus has been shown to be able to reliably place species into higher taxonomic levels while also being able to discriminate between closely related species (Herbert et al., 2003). Bone samples from six known vertebrate species were tested using the single-tube bone processing method, and a section of the COI gene was amplified, sequenced, and analyzed.

A BLAST search was used to compare the COI locus of each vertebrae sample to the database. Table 2 summarizes the BLAST search results. As with the previous BLAST search, each sample returned an E-value of zero, which is inversely proportional to the score value (over

1,000 for each sample). Additionally, the query cover percentage for all samples was 99% or higher. The query cover is the percentage of the query sequence (the test samples) that overlaps with the comparison reference sample. While this percentage alone does not guarantee a match

(given that it only states how much of the query sequence matches the reference database sequence), it, along with the zero E-value, high score value, and percent identity greater than

99%, indicates a positive match.

The use of the maceration method on the six known species samples resulted in positive identifications of all species, suggesting that the enzyme-based maceration method used in this study can be applied to species identification purposes.

45

Table 2. Summary of the sequence identification results at COI locus using GenBank BLAST of vertebrate samples

Species Alignment Query E- Percent Sample Score Identified Length (bp) Cover (%) Value Identity (%) Bovine Bos taurus 666 1230 100 0 100

Swine Sus scrofa 667 1229 100 0 100

Chicken Gallus gallus 678 1253 100 0 100

Anas Duck 662 1223 100 0 100 platyrhynchos

Sheep Ovis aries 672 1240 99 0 100

Fish Lutjanus peru 642 1181 100 0 99.84

Human Samples

To assess if this method could be applied to human samples, a small trial (n=2) using previously collected aged human bones, was carried out using mtDNA sequencing of the hypervariable regions. Figure 10 shows a representative human rib bone that was processed. A specific area of the mitochondrial genome known as the displacement loop (D-loop), or control region, is used routinely to sequence human mtDNA. The D-loop is used for transcription and replication but does not actually code for any gene products. The D-loop contains the most polymorphic areas of mtDNA, known as the hypervariable regions--specifically HVI (342 bp),

HVII (268 bp), and HVIII (438-574 bp) (Budowle, Allard, Wilson & Chakraborty, 2003). HVI and HVII demonstrate high nucleotide variability and, therefore, can be used as a means of identifying an individual. However, one caveat, as with all mtDNA analysis, is that differences in nucleotide sequence are observed only between individuals of different maternal lines (Butler, 46

2011). HVI and HVII are where the most variation from the revised Cambridge Reference

Sequence (rCRS) is found (Edson & Christensen, 2013).

Figure 10. A human bone fragment used in this study. A representative human rib bone (approximately 50 years post-mortem) is shown. .

47

Human mtDNA analysis was performed on two bone samples by sequencing the HVI region. Sequences that were of similar quality were obtained from the aged human bones after processing (see Materials and Methods); one representative electropherogram is shown (Figure

11). As mentioned previously, an ISFG hallmark of a mixture in mtDNA analysis is overlapping peaks in the electropherogram. No such indications of a mixture were observed after processing with the proposed methodology. The human mtDNA sequence from one bone sample can be seen in comparison to the rCRS in Figure 12. The rCRS is used as a reference standard where the sequence being tested is compared directly with the rCRS. Only the differences between the aligned sample and the rCRS are noted. The sequenced human bone sample had four bases that differed from the rCRS and are noted in Figure 12. These results demonstrate that the enzyme- based liquid method used can successfully macerate bone, which can then be sequenced for purposes such as DNA identification.

48

Figure 11. mtDNA sequencing results from human bones processed in this study. A representative electropherogram of HVI region is shown.

49

Figure 12. The sequence alignment of DNA contig sequences at human mitochondrial HVI locus. The contig sequence was obtained by grouping forward and reverse sequences together to produce a consensus. The sequences were aligned with the revised Cambridge Reference Sequence (rCRS). *The HVI mitotype of the sample tested was: 16092C, 16223T, 16311C and 16362C. 50

Conclusion

The incubation studies and microscopic data for the proposed maceration method demonstrated that cancellous bone exhibited evidence of degradation, which suggests the method is effective in removing surface materials prior to DNA isolation. Samples treated with the maceration method resulted in ample DNA for mtDNA analysis. Although the quantity of DNA recovered from untreated samples was higher than that of the samples treated with the maceration method, there were no significant differences in the yields obtained across all samples tested based upon ANOVA analysis. These results are similar to those obtained in earlier studies (Hochmeister et al., 1991; Li & Liriano, 2011). It is possible that the bone degradation resulting from the incubation in proteinase K, EDTA, and lauryl-sarcosinate may have removed some bone material near the surface that contained DNA.

The mtDNA sequencing of the treated Sus scrofa samples in order to evaluate the quality of the amplified cytb locus resulted in electropherograms with no evidence of adverse effects or contamination from the enzyme treatment. The quality of the treated samples also showed no differences as compared to the control samples. BLAST searches using the experimental mtDNA sequences resulted in scores (>900) that indicated a high similarity between the query sequences and the reference sequences found in the BLAST database. The E-values (0) indicated how many times the query sequences could be found by chance in the database. The use of the enzyme-based maceration method resulted in high quality DNA, which made it possible to correctly identify the Sus scrofa samples. The animal species identification experiment in this study was intended to assess the proposed maceration method’s applicability to real-life animal identification scenarios. Whether used for taxonomic or forensic cases, species identification testing is an important field. Having a more efficient, less expensive 51 method with a lower risk of contamination and possible high-throughput testing capabilities would be greatly beneficial. The testing of animal samples resulted in a 99% or higher species identification rating, which supports the proposed method’s ability to accurately identify a range of animal species. However, the species used in this study are a fraction of the possible species relevant to taxonomic and forensic cases. Further studies using a larger range of species are necessary.

The human samples in this study were used as a preliminary test to determine the method’s potential use in human testing. When compared to the rCRS, the human rib samples showed no observable contamination. The data suggests that using the enzyme maceration method may be a successful aid in both the recovery and species identification of human samples; however, further studies experimenting with different types of bone, bones found in different environmental conditions, and of varying ages are needed.

The use of enzyme-based methods for the preparation of bone samples has been described in several publications. Previous studies have shown that the use of detergents or proteases can successfully remove material from bone surfaces (Eriksen et al., 2013; Simonsen,

Rasmussen, Mathisen, Peterson & Borup 2011; Uhre et al., 2015). The results of this enzyme- based maceration method are in line with other studies using enzyme techniques to remove surface material; however, to best of this author’s knowledge, this study is the first processing method for cancellous bone that uses this particular combination of proteinase K, 0.5 M EDTA, and 1% lauryl-sarcosinate buffer for the purposes of maceration, as well as the first to introduce a single-tube testing procedure. The maceration method’s combination of chemicals can function beyond just maceration and can also be suitable for bone samples undergoing DNA isolation.

The results of this study demonstrate that samples treated with the proteinase K, EDTA, and 52 lauryl-sarcosinate maceration solution successfully had surface material removed; additionally, subsequent mtDNA testing resulted in profiles that could be used for identification purposes, establishing the proposed method as an alternative to commonly used manual or mechanical methods that can be destructive and increase the possibility of damaging samples, particularly samples that are mainly cancellous bone. The single-tube testing benefit of this method potentially decreases cross-contamination between samples (often resulting from bone powder dust that is released during processing) and sample mix-ups, in addition to allowing for the possibility of high-throughput testing, which would be especially helpful for laboratories that deal with many bone samples or mass fatality events.

53

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